Peptide fragment condensation and cyclisation using a subtilisin variant with improved synthesis over hydrolysis ratio

ABSTRACT

The invention relates to a method for enzymatically synthesizing an (oligo)peptide, comprising coupling (a) an (oligo)peptide C-terminal ester or thioester and (b) an (oligo)peptide nucleophile having an N-terminally unprotected amine, wherein the coupling is carried out in a fluid comprising water, and wherein the coupling is catalyzed by a subtilisin BPN′ variant or a homologue thereof, which comprises the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequence thereof: a deletion of the amino acids corresponding to positions 75-83; a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221 selenocysteine; preferably a mutation at the amino acid position corresponding to P225 wherein the amino acid positions are defined according to the sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2. Further, the invention relates to an enzyme suitable for use as a catalyst in a method of the invention.

The invention relates to a method for enzymatically synthesising an(oligo)peptide (i.e. a peptide, in particular oligopeptide), to anenzyme suitable for catalyzing said synthesis, to a host cell capable offunctionally expressing said enzyme and to a method for preparing saidenzyme.

Peptides, in particular oligopeptides have many applications, forinstance as pharmaceutical, food or feed ingredient, or cosmeticingredient.

Methods for synthesizing (oligo)peptides are generally known in the art.

Oligopeptides can be chemically synthesized in a stepwise fashion insolution or on the solid phase via highly optimized processes. However,peptides longer than 10-15 amino acids are often very difficult tosynthesize due to side reactions and as a consequence purification istroublesome. Therefore, peptides longer than 10 amino acids are oftensynthesized by a combination of solid-phase synthesis of side-chainprotected oligopeptide fragments which are subsequently chemicallycondensed in solution, e.g. as in a 10+10 condensation to make aoligopeptide of 20 amino acids. The major drawback of chemicalside-chain protected oligopeptide fragment condensation is that uponactivation of the C-terminal amino acid residue of the acyl donorracemisation occurs. In contrast, enzyme-catalyzed peptide couplings arecompletely devoid of racemisation and have several other advantages overchemical peptide synthesis such as the absence of side reactions on theside-chain functionalities. For industrial application, an enzymaticpeptide synthesis concept based on a kinetic approach, i.e. using anacyl donor C-terminal ester is most attractive (see for instance N.Sewald and H.-D. Jakubke, in: “Peptides: Chemistry and Biology”, 1^(st)reprint, Ed. Wiley-VCH Verlag GmbH, Weinheim 2002).

Chemo-enzymatic peptide synthesis can entail the enzymatic coupling ofoligopeptide fragments which have individually been synthesized usingchemical synthesis, fermentation, or by a combination of chemical andenzymatic coupling steps. Some reports have been published on theenzymatic condensation of oligopeptide fragments in aqueous solution(Kumaran et al. Protein Science, 2000, 9, 734; Bjorup et al. Bioorg.Med. Chem. 1998, 6, 891; Homandberg et al. Biochemistry, 1981, 21, 3387;Komoriya et al. Int. J. Pep. Prot. Res. 1980, 16, 433). However, a majordrawback of such enzymatic oligopeptide fragment condensation in aqueoussolution is that simultaneous hydrolysis of the peptide bonds within theoligopeptide fragments and of the C-terminal ester functionality takesplace leading to low yields and many side products.

Proteases have hitherto mainly been produced commercially for hydrolyticapplication, e.g. in cleaning, where peptide bonds are hydrolysed by theproteases. A typical example are the subtilisins, which form an enzymeclass with considerable importance for their use as detergents.Therefore, subtilisins have been the subject of numerous proteinengineering studies. subtilisins have also been used for the synthesisof oligopeptides, which was, however, almost always accompanied byhydrolytic side-reactions to a significant extent. It was found by Wellset al. (U.S. Pat. No. 5,403,737) that the condensation of oligopeptidesin aqueous solution could be significantly improved by altering theactive site of subtilisin BPN′, a subtilisin from B. amyloliquefaciens(SEQUENCE ID NO: 2). When two mutations were introduced, i.e. S221C andP225A, a subtilisin BPN′ variant called subtiligase was obtained havinga 500-fold increased synthesis over hydrolysis ratio (S/H ratio) ascompared to wild-type subtilisin BPN′. However, the average ligatingyield was around 66% and hydrolysis of the oligopeptide acyl donorC-terminal ester was still substantial (Wells et al. Science, 1994, 266,243). Most often, 10 equivalents of oligopeptide acyl donor C-terminalester was used to obtain a decent reaction yield. Another drawback ofsubtiligase was the poor stability against organic co-solvents that arerequired to solubilize the oligopeptide fragments, against enhancedtemperature and against denaturating agents, which are often needed forsuccessful oligopeptide condensation. Therefore, Wells et al. added fiveadditional mutations to subtiligase, i.e. M50F, N76D, N109S, K213R andN218S, to make the enzyme more stable (Proc. Natl. Acad. Sci. USA, 1994,91, 12544). The new mutant called stabiligase appeared moderately moreresistant to sodium dodecasulfate and guanidinium hydrochloride, buthydrolysis was still a major side reaction. For instance an oligopeptidecarboxyamidomethyl-ester (Cam-ester) was ligated to an oligopeptideamine using stabiligase in a yield of 44%. In this example, 10equivalents of the oligopeptide C-terminal ester were used and thus,9.56 equivalents of the oligopeptide C-terminal ester were hydrolyzed atthe C-terminal ester functionality and only 0.44 equivalents ligated tothe oligopeptide amine to form the product. Clearly, there is a need foran improved enzyme with a higher S/H ratio to make the oligopeptidecondensation reaction an economically viable process. Probably for thisreason, the past 20 years subtiligase nor stabiligase have beenindustrially applied, to the best of the inventors knowledge.

Another aspect of subtilisin BPN′ that has received attention is theincrease of the stability of the enzyme for its use as detergent (i.e.for the hydrolysis of peptide bonds) at higher temperatures and/or inthe presence of metal chelators. A typical example of such study wasdisclosed by Bryan et al. who engineered a subtilisin BPN′ variantlacking a high affinity Ca²⁺ binding site (WO02/22796). The highaffinity Ca²⁺ binding site in subtilisin BPN′ is made up by a loopcomprising amino acids 74-82 and the amino acids Gln2 (Q2) and Asp41(D41). Comparison of the 3D structure of subtilisin BPN′ with thestructure of homologous subtilisins shows that the high affinity Ca²⁺binding site is highly conserved. This binding site is important fortheir stability in known subtilisins. Stripping of the Ca²⁺ ion by forinstance metal chelators leads to unfolding and thus inactivation of theknown subtilisins. When Bryan et al. deleted amino acids 75-83 (Δ 75-83)of subtilisin BPN′ and additionally implemented the mutations Q2K, S3C,P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C,N212G, Y217L, N218S, T254A and Q271E, a subtilisin BPN′ variant wasobtained (called BS149, also known as Sbt149) which lacks the Ca²⁺binding domain and has a greatly improved stability against metalchelators (1000×). However, this enzyme cannot be used for peptidefragment condensation in aqueous solution since it is onlyhydrolytically active.

It is an object of the present invention to provide an enzymatic methodfor preparing an (oligo)peptide by condensation of a first and a second(oligo)peptide fragment or by cyclisation of an (oligo)peptide that canserve as an alternative to known methods of preparing (oligo)peptides.There is a need for alternative methods in general, in particular inorder to broaden the palette of tools for making specific(oligo)peptides.

In particular, it is an object to provide an enzymatic method forpreparing an (oligo)peptide by condensation of a first and a second(oligo)peptide fragment or by cyclisation of an (oligo)peptide, whereinan enzyme is used having an improved S/H ratio and stability compared tosubtilisin BPN′, at least under certain reaction conditions.

Further, it is an object to provide an enzymatic method for preparing an(oligo)peptide by condensation of a first and a second (oligo)peptidefragment or by cyclisation of an (oligo)peptide, wherein an enzyme isused having an improved stability compared to subtiligase, in particularan improved stability and S/H ratio compared to subtiligase.

It is yet a further object of the invention to provide the coupling of a(oligo)peptide to a protein. It is in particular a challenge to provideenzymatic methodology that allows coupling of a peptide with a protein,in particular due to the added complexity of a protein'sthree-dimensional structure.

It is yet a further object to provide a novel subtilisin BPN′ variantcapable of catalyzing the condensation of two (oligo)peptides or ofcyclisation of an (oligo)peptide, in particular such an enzyme having animproved property, such as an improved synthesis over hydrolysis ratioratio and/or improved stability, compared to known enzymes suitable tocatalyse such condensation, such as subtilisin BPN′ and/or subtiligase,at least under certain reaction conditions.

One or more other objects that may be subject of the invention followfrom the description below.

It has now surprisingly been found that it is possible to provide asubtilisin BPN′ variant wherein the calcium binding domain at thepositions corresponding to amino acids 75-83 has been inactivated,namely by deletion, that has catalytic activity with respect to thecondensation of two (oligo)peptide fragments or the cyclisation of apeptide, and in particular to provide such a variant that has animproved S/H ratio compared to subtilisin BPN′ and/or subtiligase, byproviding a subtilisin BPN′ variant that has a specific mutation,preferably a specific combination of mutations, in addition to thedeletion of the amino acids corresponding to positions 75 to 83.

Accordingly, the present invention relates to a method for enzymaticallysynthesizing an (oligo)peptide, comprising coupling (a) an(oligo)peptide C-terminal ester or thioester and (b) an (oligo)peptidenucleophile having an N-terminally unprotected amine,

-   -   wherein the coupling is carried out in a fluid comprising water,        and    -   wherein the coupling is catalyzed by a subtilisin BPN′ variant        or a homologue thereof, which comprises the following mutations        compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or        a homologue sequence thereof:

-   i) a deletion of the amino acids corresponding to positions 75-83;

-   ii) a mutation at the amino acid position corresponding to S221, the    mutation being S221C or S221selenocysteine (S221U);

-   iii) preferably, a mutation at the amino acid position corresponding    to P225;    -   wherein the amino acid positions are defined according to the        sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2.

Further, the invention relates to a method for enzymaticallysynthesizing a cyclic (oligo)peptide of at least 12 amino acids,comprising subjecting an (oligo)peptide C-terminal ester or thioesterhaving an N-terminally unprotected amine to a cyclisation step

-   -   wherein said cyclization is carried out in a fluid comprising        water, and    -   wherein the cyclization is catalyzed by a subtilisin BPN′        variant or a homologue thereof, which comprises the following        mutations compared to subtilisin BPN′ represented by SEQUENCE ID        NO: 2 or a homologue sequence thereof:

-   i) a deletion of the amino acids corresponding to positions 75-83;

-   ii) a mutation at the amino acid position corresponding to S221, the    mutation being S221C or S221selenocysteine;

-   iii) preferably, a mutation at the amino acid position corresponding    to P225;    -   wherein the amino acid positions are defined according to the        sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2.

Further, the invention relates to an enzyme, which enzyme is asubtilisin BPN′ variant or homologue thereof, comprising the followingmutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2or homologue sequence thereof:

-   -   i) a deletion of the amino acids corresponding to positions        75-83;    -   a mutation at the amino acid position corresponding to S221, the        mutation being S221C or S221selenocysteine;    -   ii) a mutation at the amino acid position corresponding to P225;    -   in which the amino acid positions are defined according to the        sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2.

Further, the invention relates to a recombinant method for preparing theenzyme according to the invention, said method comprising:

-   -   a) providing a recombinant host cell functionally expressing a        gene encoding the enzyme;    -   b) culturing said host cell under conditions which provide for        the expression of the enzymatically active enzyme; and    -   c) recovering the expressed enzyme from said microbial host.

Further, the invention relates to a recombinant polynucleotidecomprising a sequence which encodes for an enzyme according to theinvention.

Further, the invention relates to a host cell, comprising apolynucleotide according to the invention. The host cell is capable offunctionally expressing the enzyme of the invention.

Further, the invention relates to the use of an enzyme according to theinvention as a catalyst. Such use generally comprises contacting one ormore substrates (reactants) in the presence of the enzyme underconditions wherein the enzyme catalyses a chemical reaction wherein thesubstrate(s) participate(s). The enzyme has been found particularlyuseful as a catalyst in peptide synthesis. It is in particularcontemplated that an enzyme of the invention is useful for catalyzingreactions of which known subtilisins are known to be catalyticallyactive. In an embodiment, the synthesised peptide is a protein. In anembodiment the synthesised peptide is an oligopeptide. In a furtherembodiment the synthesised peptide is composed of at least 201 aminoacid units.

The invention provides a useful alternative to known methods ofpreparing (oligo)peptides, including proteins extended with an(oligo)peptide.

Moreover, it has surprisingly been found possible with a method of theinvention to enzymatically condense two (oligo)peptide fragments or tocyclize an (oligo)peptide in a liquid comprising water with a highsynthesis over hydrolysis ratio. The method of the invention isadvantageous in that it offers the possibility for coupling variousoligopeptide fragments in aqueous solution in high yield withoutsubstantial hydrolytic side reactions. Such surprising finding isillustrated by the Examples, which show that a method of the inventionis not only suitable to synthesise (oligo)peptides that lack a secondaryand tertiary protein structure, but also allows coupling two peptidefragments wherein at least one of the fragments is a protein, therebysynthesizing an (elongated) protein provided with an additional sequenceof amino acid units. It has been found possible to synthesise suchprotein whilst maintaining a secondary and tertiary structure of theprotein.

For the purpose of this invention, with “synthesis over hydrolysisratio” (S/H ratio) is meant the amount of enzymatically synthesised(oligo)peptide product divided by the amount of (oligo)peptideC-terminal ester or thioester of which the ester or thioester group hasbeen hydrolysed.

The value of the S/H ratio of an enzyme of the invention depends onvarious factors, for instance the nature of the substrates (the aminoacid sequences of the (oligo)peptide C-terminal ester or thioester andof the (oligo)peptide nucleophile) and reaction conditions (e.g.temperature, pH, concentration of the peptide fragments, enzymeconcentration). As shown in the Examples, it was found though that undervarious reaction conditions and for various substrates the S/H ratio washigher than for known subtilisins, such as subtiligase and subtilisinBPN′. Thus, it is contemplated that the S/H ratio of an enzyme accordingto the invention in general has a significantly higher S/H ratio thansubtiligase and subtilisin BPN′, when tested under the same reactionconditions and using the same substrates, and in particular it iscontemplated that an enzyme of the invention has a significantly higherS/H ratio under the conditions used in Example 1 (100 mM phosphatebuffer, pH 8.0, temperature about 20° C., concentration of(oligo)peptide C-terminal ester 0.83 mM, concentration of (oligo)peptidenucleophile 3.33 mM, enzyme concentration 5.5 mg/L) or one or more ofthe other examples. Thus, in particular, the invention relates to asubtilisin BPN′ variant or homologue thereof wherein the S/H ratio ofthe subtilisin BPN′ variant or homologue thereof divided by the S/Hratio of subtiligase—at least under the conditions described in Example1 or one or more of the other Examples—is more than 1, preferably 2 ormore, in particular 5 or more. The upper value of this quotient is notcritical; in practice it may e.g. be 100 or less, in particular 20 orless.

The S/H ratio of the subtilisin BPN′ variant or homologue thereof of theinvention divided by the S/H ratio of subtilisin BPN′—at least under theconditions described in Example 1 or one or more of the otherExamples—is usually more than 100, preferably 250 or more, morepreferably 500 or more, in particular 1000 or more. The upper value ofthis quotient is not critical; The S/H ratio of subtilisin BPN′ at leastunder the reaction conditions specified herein is generally very low, itmay be even zero (no detectible synthesis). Thus, the S/H ratio of thesubtilisin BPN′ variant or homologue thereof of the invention divided bythe S/H ratio of subtilisin BPN′ may approximate infinity. In apotential circumstance wherein subtilisin BPN′ has substantial ligase orcyclase activity, the inventors consider that the S/H ratio of thesubtilisin BPN′ variant or homologue thereof of the invention divided bythe S/H ratio of subtilisin BPN′ is also high, e.g. up to 100 000, inparticular up to 25 000, more in particular up to 10 000.

Further, using a method of the invention, the (oligo)peptide product isvery easy to purify from the reaction mixture because only littlehydrolytic by-products are formed.

Another advantage of the invention is that, due to the improved S/Hratio, a small or no excess of the (oligo)peptide C-terminal ester orthioester or of the (oligo)peptide nucleophile is needed to reach a highyield (>80%) in the condensation reaction. Accordingly, in anadvantageous embodiment an (oligo)peptide C-terminal ester or thioesterand an (oligo)peptide nucleophile are contacted in a small excess of oneof said (oligo)peptide fragments or in an about stoichiometric ratioalthough a larger excess of one over the other may be used, as describedbelow.

As illustrated by the Examples, an enzyme according to the invention isalso advantageous in that it allows the synthesis of a cyclic(oligo)peptide with significantly higher yield than with subtiligase(78% versus 61% for subtiligase). Cyclic (oligo)peptides are aparticularly interesting class of peptides since they are often morepotent due to their more constrained three dimensional structure andhigher resistance to proteolysis.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show enzymatic activity respectively S/H ratio ofdifferent enzymes of the invention, compared to subtiligase; allindicated mutations on M222, Y104, I107 and/or L135 are additional tothose of BS149-DM. The name ‘BS149-DM’ is used herein for the subtilisinBPN′ variant which has the following mutations compared to subtilisinBPN′ (SEQUENCE ID NO 2): a deletion of the amino acids 75-83 (Δ75-83)S221C, P225A, Y217L, Q2K, S3C, P5S, S9A, I13L, K43N, M50F, A73L, E156S,G166S, G169A, S188P, Q206C, N212G, N218S, T254A and Q271E.

FIGS. 2A and 2B show activity respectively S/H ratio of differentBS149-DM+M222P+L217 mutants; all indicated mutations on L217 areadditional to those of BS149-DM+M222P.

FIG. 3A: The P4 pocket specificity of BS149-DM and BS149-DM+Y104mutants.

FIG. 3B: The P4 pocket specificity of BS149-DM and BS149-DM+1107mutants.

FIG. 3C: The P4 pocket specificity of BS149-DM and BS149-DM+L135 mutants

FIG. 4A: The P1′ pocket specificity of BS149-DM and BS149-DM+M222A,M222E and M222Q mutants

FIG. 4B: The P2′ pocket specificity of BS149-DM and BS149-DM+M222A,M222E and M222Q mutants

FIG. 4C: The P1′ pocket specificity of BS149-DM and BS149-DM+M222G,M222N and M222P mutants

FIG. 4D: The P2′ pocket specificity of BS149-DM and BS149-DM+M222G,M222N and M222P mutants

FIG. 5A: The P1′ pocket specificity of BS149-DM+M222P+L217N, L217T andL217E mutants

FIG. 5B: The P2′ pocket specificity of BS149-DM+M222P+L217N, L217T andL217E mutants

FIG. 5C: The P1′ pocket specificity of BS149-DM+M222P+L2171, L217V andL217A mutants

FIG. 5D: The P2′ pocket specificity of BS149-DM+M222P+L2171, L217V andL217A mutants

FIG. 5E: The P1′ pocket specificity of BS149-DM+M222P+L217M, L217K andL217Q mutants

FIG. 5F: The P2′ pocket specificity of BS149-DM+M222P+L217M, L217K andL217Q mutants

FIG. 5G: The P1′ pocket specificity of BS149-DM+M222P+L217S, L217G andL217Y mutants

FIG. 5H: The P2′ pocket specificity of BS149-DM+M222P+L217S, L217G andL217Y mutants

FIG. 5I: The P1′ pocket specificity of BS149-DM+M222P+L217F, L217H andL217W mutants

FIG. 5J: The P2′ pocket specificity of BS149-DM+M222P+L217F, L217H andL217W mutants

FIG. 5K: The P1′ pocket specificity of BS149-DM+M222P+L217R, L217C,L217D and L217P mutants

FIG. 5L: The P2′ pocket specificity of BS149-DM+M222P+L217R, L217C,L217D and L217P mutants

FIG. 6A: The P1′ pocket substrate specificity of BS149-DM+M222G+L217N,L217T and L217E mutants

FIG. 6B: The P1′ pocket substrate specificity of BS149-DM+M222G+L2171,L217V and L217A mutants

FIG. 6C: The P1′ pocket substrate specificity of BS149-DM+M222G+L217M,L217K and L217Q mutants

FIG. 6D: The P1′ pocket substrate specificity of BS149-DM+M222G+L217S,L217G and L217Y mutants

FIG. 6E: The P1′ pocket substrate specificity of BS149-DM+M222G+L217F,L217H and L217R mutants

FIG. 6F: The P1′ pocket substrate specificity of BS149-DM+M222G+L217C,L217D and L217P mutants

FIG. 7A: The P1′ pocket specificity of BS149-DM, BS149-DM+M222G andBS149-DM+I107V+M222G mutants

FIG. 7B: The P2′ pocket specificity of BS149-DM, BS149-DM+M222G andBS149-DM+I107V+M222G mutants

FIG. 7C: The P4 pocket specificity of BS149-DM, BS149-DM+I1O7V andBS149-DM+I107V+M222G mutants

FIG. 8: S/H ratio of BS149-DM+M222G mutant at different pH values

FIG. 9A: S/H ratio of BS149-DM+M222G mutant using differentconcentrations of acyl donor and H-Glu-Leu-Arg-NH₂ nucleophile

FIG. 9B: S/H ratio of BS149-DM+M222G mutant using differentconcentrations of acyl donor and H-Ala-Leu-Arg-NH₂ nucleophile

FIG. 10: S/H ratio of different enzymes of the invention used for(oligo)peptide cyclization, compared to subtiligase.

FIG. 11: S/H ratio of BS149-DM+M222G mutant used for (oligo)peptidecyclization at different pH values

FIG. 12: B. subtilis/E. coli shuttle vector pBE-S with BS149-DM gene(pBES DNA-BS149-DM HIStag)

FIG. 13: B. subtilis/E. coli shuttle vector PBS42-S5 with Subtiligasegene

FIG. 14: list of subtilisins that may be used as a template for theprovision of homologues of subtilisin BPN′ variants of the invention andthe alignment of the sequence segments containing the Ca2+ binding loopwith the corresponding loop in subtilisin BPN′ (SEQ ID NO 2) and thedeletion of the loop in BS149-DM (SEQ ID NO 5).

The polynucleotide of the invention can be in either single ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides. A polynucleotide can be full-length ora subsequence of a native or heterologous structural or regulatory gene.Unless otherwise indicated, the term includes reference to the specifiedsequence as well as the complementary sequence thereof. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including among other things, simple and complex cells.

The recombinant polynucleotide of the invention is typically synthetic.The invention in particular extends to DNA or RNA isolated from anyorganism. In a specific embodiment, the invention extends to a host cellcomprising recombinant DNA according to the invention. The host cell istypically transgenic.

The term “recombinant” as used herein, refers to a polynucleotide or acell containing the polynucleotide, which is the result of one or moregenetic modifications using (a) recombinant DNA technique(s) and/or(an)other mutagenic technique(s). In particular a recombinant cell maycomprise a polynucleotide not present in a corresponding wild-type cell,which polynucleotide has been introduced into that cell usingrecombinant DNA techniques (a transgenic cell), or which polynucleotidenot present in said wild-type cell is the result of one or moremutations—for example using recombinant DNA techniques or anothermutagenesis technique such as UV-irradiation—in a polynucleotidesequence present in said wild-type cell (such as a gene encoding awild-type polypeptide, such as an enzyme) or wherein the polynucleotidesequence of a gene has been modified to target the polypeptide product(encoding it) towards another cellular compartment. Further, the term“recombinant (cell)” in particular relates to a strain (cell) from whichDNA sequences have been removed using recombinant DNA techniques.

In particular, the introduction of a mutation into a polynucleotidesequence to exchange one nucleotide for another nucleotide may beaccomplished by site-directed mutagenesis using any of the methods knownin the art. Furthermore mutated genes may be obtained by gene synthesiswhich apart from the introduction of changes at the amino acid level,may also be used to optimize the coding sequence to improvetranscription and translation (R. Carlson, Nature Biotechnology, 2009,27, 1091; E. Angov et al., PLoS ONE 2008, 3(5): e2189.

The term “transgenic cell ” as used herein, refers to a strain (cell)containing a polynucleotide not naturally occurring in that strain(cell) and which has been introduced into that strain (cell) usingrecombinant DNA techniques, i.e. a recombinant cell).

The term “or” as used herein is defined as “and/or” unless it isspecified otherwise or it follows from the context that it means ‘either. . . or . . . ’.

The term “a” or “an” as used herein is defined as “at least one” unlessit is specified otherwise or it follows from the context that it shouldrefer to the singular only.

When referring to a noun (e.g. a compound, an additive, etc.) in thesingular, the plural is meant to be included, unless it follows from thecontext that it should refer to the singular only.

For the purpose of this invention, with “peptides” is meant any chaincomposed of two or more amino acids. Thus, peptides are generally amidesat least conceptually composed of two or more amino carboxylic acidmolecules (i.e. amino acids) by formation of a covalent bond from thecarbonyl carbon of one to the nitrogen atom of another with formal lossof water. The term is usually applied to structures formed fromalpha-amino acids. A peptide may be linear, branched or cyclic. Apeptide can have a single chain composed of two or more amino acids or apeptide can have a plurality of chains. In the case a peptide iscomposed of two or more chains, each chain generally is composed ofthree or more amino acid molecules. The amino acid sequence of a peptideis referred to as the primary structure.

In an embodiment, the peptide is essentially free of a secondarystructure and essentially free of a tertiairy structure.

In a further embodiment, the peptide has a secondary structure.Secondary structures are generally highly regular local sub-structures,such as alpha-helices and beta-sheets (or beta-strands), by interactionsbetween the individual amino acids and the peptide backbone.

In an embodiment, the peptide (or plurality of peptides) has a tertiarystructure. Tertiary structures are generally formed by multipleinteractions, among others hydrogen bonding, hydrophobic interactions,van der Waals interactions, ionic interactions and disulphide bonds. Thesecondary structure can also contribute to the tertiary structure. Thetertiary structure provides a three-dimensional shape (which isessentially fixed in a stable environment, such as in the absence of achange in temperature and in the absence of a change in the mediumwherein the peptide is present, etc.). As the skilled person knows, thetertiary structure is different from a random coil peptide chain lackingany fixed three-dimensional structure. Proteins are (oligo)peptideshaving a tertiary structure. A well known example of tertiary structureis the globular structure of globular proteins. In an embodiment, theprotein is a protein for target delivery of a pharmaceutically active(oligo)peptide to a specific site, e.g. to a tumour or to organ tissue.Well known examples of proteins, suitable for such purpose, areimmunoglobulins or parts thereof, such as an antigen-binding fragment(Fab) of an immunoglobulin. Immuglobulins coupled to a pharmaceuticallyactive (oligo)peptide can thus be used to more efficiently deliver apharmaceutically active (oligo)peptide to a target, e.g. tumor tissue ororgan tissue, that contain an antigen for the immunoglobulin. In anembodiment, the protein is a protein suitable to increase the half-lifeof an (oligo)peptide in a living organism, in particular the bloodplasma half-life. Albumins are examples of proteins that can be coupledto an (oligo)peptide to increase the half-life.

Disulphide bonds (disulphide bridges) are typically bonds between twocysteine units (formed by oxidation). Thus, two amino acids in a samepeptide chain (amino acid sequence) can be covalently bound, also ifthey are not adjacent amino acids in the amino acid sequence. Also, adisulphide bond between a first cysteine of a first peptide chain and asecond cysteine of a second peptide chain, which may have the same or adifferent amino acid sequence, can be formed to form a peptide. Suchpeptide comprises more than one peptide chain. An example of a peptidecomposed of more than one peptide chain, wherein the different chainsare bound via a disulphide bond is insulin. Other bonds to joindifferent peptide chains are generally known in the art.

In an embodiment, the (oligo)peptide essentially consists of amino acidunits. In a further embodiment, the (oligo)peptide essentially consistsof amino acid units and protective groups. In an embodiment, the peptideis a conjugate of a peptide chain of two or more amino acids and anothermolecule, in particular a carobohydrate or a lipid. These peptides arecalled glycopeptides and lipopeptides respectively. In a furtherembodiment, the peptide conjugate is a conjugate of two or more aminoacids and an imaging agent, such as a fluorescent, phosphorescent,chromogenic or radioactive group. The peptide conjugate may also containa chelation agent or toxin.

Typically, a peptide—which term includes oligopeptides, proteins andpeptide conjugates—comprises up to about 35 000 amino acid units, inparticular 3-20 000 amino acid units, more in particular 4-5 000 aminoacid units, preferably 5-1000 amino acid units. In a specificallypreferred embodiment the peptide comprises 500 amino acid units or less,in particular 200 or less, more in particular 100 or less In aspecifically preferred embodiment, the peptide comprises at least 10amino acid units, more specifically at least 15 amino acids, at least 25amino acids or at least 40 amino acids.

With “oligopeptides” is meant within the context of the invention, apeptide composed of 2-200 amino acid units, in particular composed of5-100 amino acid units, more in particular composed of 10-50 amino acidunits.

The term “(oligo)peptide” is used herein as a short-hand for the phrase“peptides, in particular oligopeptides”.

The (oligo)peptide that is synthesized may be linear, branched orcyclic. Good results have been achieved with the synthesis of a linearor cyclic oligopeptide. Further good results have been achieved in thesynthesis of a peptide having more than 200 amino acid units, e.g. ofabout 800 amino acid units. Thus, the peptide can have at least 250amino acid units or at least 400 amino acid units. Further, good resultshave been achieved with the coupling of a peptide fragment to a protein,such as insulin, whilst maintaining a secondary and tertiary proteinstructure. The protein can have 200 or less amino acid units or can havemore than 201 amino acid units.

The non-cyclic (oligo)peptides are synthesized from a first(oligo)peptide and a second (oligo)peptide, which are both smaller thanthe (oligo)peptide that is synthesized. The first (oligo)peptide is an(oligo)peptide C-terminal ester or thioester and the second(oligo)peptide comprises an N-terminally unprotected amine. The(oligo)peptide C-terminal ester or thioester is also referred to as an(oligo)peptide acyl donor. The second (oligo)peptide is also referred toas an (oligo)peptide nucleophile. These (oligo)peptides from which thesynthesised (oligo)peptide is formed are referred to herein as‘(oligo)peptide fragments’. These (oligo)peptide fragments can on theirturn be synthesized enzymatically from a smaller (oligo)peptide acyldonor and an (oligo)peptide nucleophile or by regular chemical solutionor solid phase peptide synthesis, known by the person skilled in theart.

For the purpose of this invention, with “peptide bond” is meant theamide bond between (i) either the alpha-amino terminus of onealpha-amino acid or the beta-amino acid terminus of one beta-amino acidand (ii) either the alpha-carboxyl terminus of one other alpha-aminoacid or the beta-carboxyl terminus of one other beta-amino acid.Preferably, the peptide bond is between the alpha-amino terminus of oneamino acid and the alpha-carboxyl terminus of another amino acid.

For the purpose of this invention, with “cyclic peptide” is meant an(oligo)peptide chain wherein the alpha-amino terminus and thealpha-carboxyl terminus of a branched or linear (oligo)peptide arelinked via a peptide bond, thereby forming a ring structure of at least12 amino acid units. The cyclic peptide is in particular composed of12-200 amino acid units, more in particular composed of 12-100 aminoacid units and preferably composed of 12-50 amino acid units.

For the purpose of this invention, with “condensation” is meant theformation of a new peptide bond between the C-terminal carboxylicfunction of an (oligo)peptide with the N-terminal amine function ofanother (oligo)peptide or of the same (oligo)peptide.

In the context of this application, the term “about” means in particulara deviation of 10% or less from the given value, more in particular 5%or less, even more in particular 3% or less.

As defined by Schechter and Berger, the active site residues inproteases, including subtilisins, are composed of contiguous pocketstermed subsites. Each subsite pocket binds to a corresponding residue inthe peptide substrate sequence, referred to here as the sequenceposition. According to this definition, amino acid residues in thesubstrate sequence are consecutively numbered outward from the cleavagesites as . . . -P4-P3-P2-P1-P1′-P2′-P3′-P4′- . . . (the scissile bond islocated between the P1 and P1′ positions), while the subsites in theactive site are correspondingly labelled as . . .-S4-S3-S2-S1-S1′-S2′-S3′-S4′-. (Schechter and Berger, Biochem BiophysRes Commun. 1967 Apr. 20; 27(2):157-62.)).

For the purpose of this invention, with “S1, S2, S3 and S4 pocket” ismeant the amino acids of a protease which interact with the amino acidsof an (oligo)peptide acyl donor. The C-terminal amino acid (1st aminoacid; P1) of the acyl donor (oligo)peptide interacts with the aminoacids in the S1 pocket of the protease. The penultimate amino acid(2^(nd) amino acid; P2) of the acyl donor (oligo)peptide interacts withthe amino acids in the S2 pocket of the protease, the third amino acid(P3) with the S3 and the fourth amino acid (P4) with the S4 pocket. TheS1-S4 binding pockets of a protease are defined by several amino acidswhich can be distant in the primary structure of the protease, but areclose in the three dimensional space. For the purpose of this invention,with S1′ and S2′ pockets are meant the amino acids of a protease whichinteract with the N-terminal amino acids of an (oligo)peptidenucleophile. The N-terminal amino acid of the (oligo)peptide nucleophileinteracts with the amino acids in the S1′ pocket of the protease. TheN-terminal penultimate amino acid of the (oligo)peptide nucleophileinteracts with the amino acids in the S2′ pocket of the protease. TheS1′ and S2′ binding pockets of a protease are defined by several aminoacids which can be distant in the primary structure of the protease, butare close in the three dimensional space.

For the purpose of this invention, with “denaturating agent” is meant anadditive which potentially can destroy the three dimensional structureof a protease, and thus, can potentially inactivate the protease.

In the context of the invention with “amino acid side-chain” is meantany proteinogenic or non-proteinogenic amino acid side-chain.

Proteinogenic amino acids are the amino acids that are encoded by thegenetic code. Proteinogenic amino acids include: alanine (Ala), valine(Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr),methionine (Met), cysteine (Cys), asparagine (Asn), glutamine (Gln),tyrosine (Tyr), tryptophan (Trp), glycine (Gly), aspartic acid (Asp),glutamic acid (Glu), histidine (His), lysine (Lys), arginine (Arg),proline (Pro) and phenylalanine (Phe). Selenocysteine (Sec, U) is anamino acid, of which the structure corresponds to cysteine, with theproviso that it contains a selenium instead of a sulphur atom.

Non-proteinogenic amino acids may in particular be selected amongstD-amino acids, L- or D-phenylglycine, DOPA(3,4-dihydroxy-L-phenylalanine), beta-amino acids,4-fluoro-phenylalanine, or C^(α)-alkylated amino acids.

The term “mutated” or “mutation” as used herein regarding proteins orpolypeptides—in particular enzymes—means that at least one amino acid inthe wild-type or naturally occurring protein or polypeptide sequence hasbeen replaced with a different amino acid, inserted into, appended to,or deleted from the sequence via mutagenesis of nucleic acids encodingthese amino acids. Mutagenesis is a well-known method in the art, andincludes, for example, site-directed mutagenesis by means of PCR or viaoligonucleotide-mediated mutagenesis as described in Sambrook et al.,Molecular Cloning-A Laboratory Manual, 2nd ed., Vol. 1-3 (1989). Theterm “mutated” or “mutation” as used herein regarding genes means thatat least one nucleotide in the nucleic acid sequence of that gene or aregulatory sequence thereof, has been replaced with a differentnucleotide, has been inserted into, has been appended to, or has beendeleted from the sequence via mutagenesis, resulting in thetranscription of a protein sequence with a qualitatively ofquantitatively altered function or resulting in the knock-out of thatgene.

In the present specification, a shorthand for denoting amino acidsubstitutions employs the single letter amino acid code of the aminoacid that is substituted, followed by the number designating where inthe protein amino acid sequence the substitution is made. This number isthe amino acid position of the wild-type amino acid sequence (generallysubtilisin BPN′ unless specified otherwise). Thus for the mutated aminoacid sequence it is the amino acid position corresponding to theposition with that number in the wild type enzyme. Due to one or moreother mutations at a lower position (additions, insertions, deletions,etc.) the actual position does not need to be the same. The skilledperson will be able to determine the corresponding positions using agenerally known alignment technique, such as NEEDLE. The number isfollowed by the single letter code of the amino acid that replaces thewild-type amino acid therein. For example, G166S denotes thesubstitution of glycine at the position corresponding to position 166 toserine. X is used to indicate any other proteinogenic amino acid thanthe amino acid to be substituted. For example, G166X denotes thesubstitution of glycine 166 to any other proteinogenic amino acid.

When referring to a compound of which stereoisomers exist, the compoundmay be any of such stereoisomers or a mixture thereof. Thus, whenreferred to, e.g., an amino acid of which enantiomers exist, the aminoacid may be the L-enantiomer, the D-enantiomer or a mixture thereof. Incase a natural stereoisomer exists, the compound is preferably a naturalstereoisomer.

The term ‘pH’ is used herein for the apparent pH, i.e. the pH asmeasured with a standard, calibrated pH electrode.

When an enzyme is mentioned with reference to an enzyme class (EC)between brackets, the enzyme class is a class wherein the enzyme isclassified or may be classified, on the basis of the Enzyme Nomenclatureprovided by the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology (NC-IUBMB), which nomenclature may befound at http://www.chem.qmul.ac.uk/iubmb/enzyme/. Other suitableenzymes that have not (yet) been classified in a specified class but maybe classified as such, are meant to be included.

Homologues typically have an intended function in common with thepolynucleotide respectively polypeptide (enzyme) of which it is ahomologue, such as encoding the same peptide respectively being capableof catalyzing the same reaction. The term homologue is also meant toinclude nucleic acid sequences (polynucleotide sequences) which differfrom another nucleic acid sequence due to the degeneracy of the geneticcode and encode the same polypeptide sequence.

Amino acid or nucleotide sequences are said to be homologous whenexhibiting a certain level of similarity. Two sequences being homologousindicate a common evolutionary origin. Whether two homologous sequencesare closely related or more distantly related is indicated by “percentidentity” or “percent similarity”, which is high or low respectively.

The terms “homology”, “percent homology”, “percent identity” or “percentsimilarity” are used interchangeably herein. For the purpose of thisinvention, it is defined here that in order to determine the percentidentity of two amino acid sequences or of two nucleic acid sequences,the complete sequences are aligned for optimal comparison purposes. Inorder to optimize the alignment between the two sequences gaps may beintroduced in any of the two sequences that are compared. Such alignmentis carried out over the full length of the sequences being compared.Alternatively, the alignment may be carried out over a shorter length,for example over about 20, about 50, about 100 or more nucleic acids oramino acids. The percentage identity is the percentage of identicalmatches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm. Theskilled person will be aware of the fact that several different computerprograms are available to align two sequences and determine the homologybetween two sequences (Kruskal, J. B. (1983) An overview of sequencecomparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, stringedits and macromolecules: the theory and practice of sequencecomparison, pp. 1-44 Addison Wesley). The percent identity between twoamino acid sequences can be determined using the Needleman and Wunschalgorithm for the alignment of two sequences. (Needleman, S. B. andWunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm alignsamino acid sequences as well as nucleotide sequences. TheNeedleman-Wunsch algorithm has been implemented in the computer programNEEDLE. For the purpose of this invention the NEEDLE program from theEMBOSS package was used (version 2.8.0 or higher, EMBOSS: The EuropeanMolecular Biology Open Software Suite (2000) Rice, P. Longden, I. andBleasby, A. Trends in Genetics 16, (6) pp 276-277,http://emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 isused for the substitution matrix. For nucleotide sequences, EDNAFULL isused. Other matrices can be specified. The optional parameters used foralignment of amino acid sequences are a gap-open penalty of 10 and a gapextension penalty of 0.5. The skilled person will appreciate that allthese different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

The homology or identity between the two aligned sequences is calculatedas follows: the number of corresponding positions in the alignmentshowing an identical amino acid in both sequences divided by the totallength of the alignment after subtraction of the total number of gaps inthe alignment. The identity defined as herein can be obtained fromNEEDLE by using the NOBRIEF option and is labelled in the output of theprogram as “longest-identity”. For purposes of the invention the levelof identity (homology) between two sequences (amino acid or nucleotide)is calculated according to the definition of “longest-identity” as canbe carried out by using the program NEEDLE.

The polypeptide sequences representing an enzyme of the presentinvention, can further be used as a “query sequence” to perform a searchagainst sequence databases, for example to identify other family membersor related sequences. Such searches can be performed using the BLASTprograms. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequencesand BLASTN for nucleotide sequences. The BLAST program uses as defaults:

-   Cost to open gap: default=5 for nucleotides/11 for proteins-   Cost to extend gap: default=2 for nucleotides/1 for proteins-   Penalty for nucleotide mismatch: default=−3-   Reward for nucleotide match: default=1-   Expect value: default=10-   Wordsize: default=11 for nucleotides/28 for megablast/3 for proteins

Furthermore the degree of local identity (homology) between the aminoacid sequence query or nucleic acid sequence query and the retrievedhomologous sequences is determined by the BLAST program. However onlythose sequence segments are compared that give a match above a certainthreshold. Accordingly the program calculates the identity only forthese matching segments. Therefore the identity calculated in this wayis referred to as local identity.

The term “homologue” is used herein in particular for polypeptides(enzymes) having a sequence identity of at least 50%, preferably atleast 60%, more preferably at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99% withthe polypeptide (enzyme) with which the homologue peptide is compared.Evidently, the sequence identity will be less than 100%. The percentageof sequence identity will depend on the number of mutations and thelength of the polypeptide with which the homologue is prepared. Inparticular, for a subtilisin BPN′ variant, the number of mutations forthe enzymes in the present invention will typically be at least 11, ofwhich at least nine mutations are deletions and at least two mutationsare replacements for another amino acid. In ‘longest identity’ alignmentthe deletions are not taken into account. This means that the sequenceidentity of an enzyme of the invention compared to subtilisin BPN′generally is 99.25% (two replacements in a polypeptide with 266 aminoacids) or less. Preferably, the sequence identity of an enzyme of theinvention compared to SEQUENCE ID NO 2, is 98% or less, more preferably96% or less, in particular 94% or less, more in particular 92% or less,or 90% or less.

“Expression” refers to the transcription of a gene into structural RNA(rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into aprotein.

As used herein, “heterologous” in reference to a nucleic acid or proteinis a nucleic acid or protein that originates from a foreign species, or,if from the same species, is substantially modified from its native formin composition and/or genomic locus by deliberate human intervention.For example, a promoter operably linked to a heterologous structuralgene is from a species different from that from which the structuralgene was derived, or, if from the same species, one or both aresubstantially modified from their original form. A heterologous proteinmay originate from a foreign species or, if from the same species, issubstantially modified from its original form by deliberate humanintervention.

The term “heterologous expression” refers to the expression ofheterologous nucleic acids in a host cell. The expression ofheterologous proteins in suitable host cell systems are well known tothose of skill in the art. The skilled person will be able to providesuitable host cells for producing an enzyme of the invention fromvarious organisms without undue burden based upon common generalknowledge and the information disclosed herein.

As used herein “promoter” is a DNA sequence that directs thetranscription of a (structural) gene. Typically, a promoter is locatedin the 5′ region of a gene, proximal to the transcriptional start siteof a (structural) gene. Promoter sequences may be constitutive,inducible or repressible. If a promoter is an inducible promoter, thenthe rate of transcription increases in response to an inducing agent.

The term “vector” as used herein, includes reference to an autosomalexpression vector and to an integration vector used for integration intothe chromosome.

The term “expression vector” refers to a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide (enzyme) ofinterest under the control of (i.e. operably linked to) additionalnucleic acid segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

“Plasmid” refers to autonomously replicating extrachromosomal DNA whichis not integrated into a microorganism's genome and is usually circularin nature.

An “integration vector” refers to a DNA molecule, linear or circular,that can be incorporated in a microorganism's genome and provides forstable inheritance of a gene encoding a polypeptide of interest. Theintegration vector generally comprises one or more segments comprising agene sequence encoding a polypeptide of interest under the control of(i.e. operably linked to) additional nucleic acid segments that providefor its transcription. Such additional segments may include promoter andterminator sequences, and one or more segments that drive theincorporation of the gene of interest into the genome of the targetcell, usually by the process of homologous recombination. Typically, theintegration vector will be one which can be transferred into the targetcell, but which has a replicon which is nonfunctional in that organism.Integration of the segment comprising the gene of interest may beselected if an appropriate marker is included within that segment.

As used herein, the term “operably linked” refers to a juxtapositionwherein the components so described are in a relationship permittingthem to function in their intended manner. A control sequence “operablylinked” to another control sequence and/or to a coding sequence isligated in such a way that transcription and/or expression of the codingsequence is achieved under conditions compatible with the controlsequence. Generally, operably linked means that the nucleic acidsequences being linked are contiguous and, where necessary to join twoprotein coding regions, contiguous and in the same reading frame.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as bacterial cells, or eukaryotic cells such asyeast, plant, insect, amphibian, or mammalian cells.

“Transformation” and “transforming”, as used herein, refers to theinsertion of an exogenous polynucleotide into a host cell, irrespectiveof the method used for the insertion, for example, direct uptake,transduction, f-mating or electroporation. The exogenous polynucleotidemay be maintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host cell genome.

For the purpose of clarity and a concise description features aredescribed herein as part of the same or separate embodiments, however,it will be appreciated that the scope of the invention may includeembodiments having combinations of all or some of the featuresdescribed.

The term ‘C-terminal protection’ is used herein to indicate that aC-terminal carboxylic group of an (oligo)peptide is provided with aprotective group, generally substantially protecting the carboxylicgroup from being coupled to an N-terminal amine group of another(oligo)peptide or of the same (oligo)peptide molecule. The C-terminalprotective group may be a t-alkyl ester group for instance a t-butylester group, which is a commonly used protective group. The C-terminalprotective group may also be a C-terminal carboxy-amide. A primarycarboxy-amide is a commonly used protective group.

The term ‘N-terminal protection’ is used herein to indicate that anN-terminal amine group of an (oligo)peptide is provided with aprotective group, generally at least substantially protecting theN-terminal amine group from being coupled to a C-terminal carboxylicgroup of another (oligo)peptide or of the same (oligo)peptide molecule.

The (oligo)peptide C-terminal ester or thioester typically is anactivated (thio)ester, i.e. it contains a carboxy ester or carboxythioester group that can take part in the enzymatic coupling reaction.In principle, any (substituted or unsubstituted) alkyl or (substitutedor unsubstituted) aryl (thio)ester can be used. Typical examples of(thio)esters which can take part in the enzymatic coupling reaction aremethyl-, ethyl, propyl-, isopropyl-, phenyl-, benzyl-,2,2,2-trichloroethyl-, 2,2,2-trifluoroethyl-, cyanomethyl- andcarboxyamidomethyl-(thio)esters.

Particularly good results have been obtained withcarboxyamidomethyl-type esters represented by the formulapeptide-(C═O)—O—CX₁X₂—C(═O)N—R₁R₂. Herein, each X₁ and X₂ independentlyrepresents a hydrogen atom or an alkyl group. Good results have beenachieved when both X₁ and X₂ are a hydrogen atom(peptide-(C═O)—O—CH₂—C(═O)N—R₁R₂). Herein R₁ represents a hydrogen atomor an alkyl group and R₂ represents a hydrogen atom or an alkyl group oran amino acid or a peptide residue with a C-terminal carboxyamide orcarboxylic acid functionality, optionally protected on the side-chainfunctionality of the amino acid or on one or more of the side-chainfunctionalities of the amino acids. Herein, each alkyl group mayindependently represent a (substituted or unsubstituted) C1-C7 alkylgroup, preferably a (substituted or unsubstituted) linear C1-C6 alkylgroup, more preferably a (substituted or unsubstituted) linear C1-C3alkyl group, and most preferably a methyl group. Good results have inparticular been achieved in a method of the invention wherein both R₁and R₂ represent a hydrogen atom or wherein R₁ represents a hydrogenatom and R₂ represents an amino acid or peptide residue with aC-terminal carboxyamide or carboxylic acid functionality, optionallyprotected on the side-chain functionality of the amino acid or on one ormore of the side-chain functionalities of the amino acids. Particularlygood results have been achieved when using the Cam-ester, when X₁, X₂,R₁ and R₂ are a hydrogen atom.

The (oligo)peptide C-terminal (thio)ester can be N-terminallyunprotected or N-terminally protected. In an embodiment, one or moreside-chain functionalities (in particular carboxyl groups, aminegroups), e.g. all side-chain functionalities, are provided with aprotecting group; in another embodiment all the side-chainfunctionalities are unprotected. In a preferred embodiment, only theside-chain functionalities of the amino acids at the P4 and P1 positionof the (oligo)peptide acyl donor and at the P1′ or P2′ position of the(oligo)peptide nucleophile (in particular hydroxy groups, carboxylgroups or amine groups) are provided with a protecting group. Suitableprotecting groups are known to the person skilled in the art. Carboxylicacid groups can for instance be protected with a cyclohexyl, benzyl orallyl group; amine functionalities can for instance be protected with anallyloxycarbonyl group or a trifluoroacetyl group.

The activated C-terminal (thio)ester group of the (oligo)peptideC-terminal (thio)ester can be synthesized using solid phase synthesis inhigh yield and purity without racemization. An additional advantage ofthe use of (thio)esters wherein R₁ represents a hydrogen atom and R₂represents an amino acid or peptide residue with a C-terminal carboxylicacid functionality, optionally protected on the side-chain functionalityof the amino acid or on one or more of the side-chain functionalities ofthe amino acids is, that their activated C-terminal ester or thioestergroup can be synthesized using the cheap and industrially available2-chlorotritylchloride resin.

The activated C-terminal (thio)ester group of the (oligo)peptideC-terminal (thio)ester can also be synthesized by fermentation using amicroorganism. A reliable method to obtain (oligo)peptide (thio)estersusing fermentation is via so-called intein expression (see for instanceE. K. Lee, Journal of Chemical Technology and Biotechnology, 2010, 9,11-18). Different intein expression systems kits are commerciallyavailable (for instance the IMPACT™ kit). Other methods for thefermentative production of (oligo)peptide (thio)esters are known in theart.

The C-terminal amino acid of the (oligo)peptide C-terminal (thio)esterand the other amino acids of the (oligo)peptide C-terminal (thio)estermay in principle be any amino acid, proteinogenic or non-proteinogenic.If the amino acid sequence of the C-terminal part of the (oligo)peptideC-terminal (thio)ester is poorly recognized by or inaccessible to thecoupling enzyme due to the amino acid preference of the coupling enzymeand/or due to the secondary or tertiary structure of the (oligo)peptide,the primary structure (amino acid sequence) may be elongated at theC-terminus. Essentially the C-terminus of the (oligo)peptide C-terminal(thio)ester is elongated with a number of amino acids to ensure goodrecognition by the enzyme and accessibility into the enzyme for theenzymatic coupling reaction. The skilled person will know how toelongate the (oligo)peptide C-terminal (thio)ester on the basis of theinformation disclosed herein and common general knowledge. Usually thenumber of amino acids for elongation is in the range of 1-10, althoughin principle it can be higher. Good results have been obtained byelongation of the (oligo)peptide C-terminal (thio)ester with 4 aminoacid residues, e.g. -Phe-Ser-Lys-Leu-(thio)ester.

In particular the (optionally N-terminal protected) (oligo)peptideC-terminal (thio)ester may be represented by a compound of Formula I.

Herein Q represents an OR or SR moiety. R may represent a (substitutedor unsubstituted) alkyl or a (substituted or unsubstituted) aryl group.

Herein P¹ stands for a hydrogen or an N-terminal protecting group.Suitable N-terminal protecting groups are those N-protecting groupswhich can be used for the synthesis of (oligo)peptides. Such groups areknown to the person skilled in the art. Examples of suitableN-protecting groups include carbamate or acyl type protecting groups,for instance ‘Cbz’ (benzyloxycarbonyl), ‘Boc’ (tert-butyloxycarbonyl),‘For’ (formyl), ‘Fmoc’ (9-fluorenylmethoxycarbonyl), ‘PhAc’ (phenacetyl)and ‘Ac’ (acetyl). The groups For, PhAc and Ac may be introduced andcleaved enzymatically using the enzymes Peptide Deformylase, PenGacylase or Acylase, respectively. Chemical cleavage methods aregenerally known in the art.

Herein, n is an integer of at least 2. n May in particular be at least3, at least 4, at least 5, at least 6, at least 7 at least 8, at least 9or at least 10. n May in particular be 100 or less, 75 or less, 50 orless, 25 or less, 20 or less 15 or less, e.g. 10 or less.

Herein, each R^(A) and each R^(B) independently represent a hydrogenatom or an organic moiety, preferably an amino acid side-chain. Thus, itis not required that R^(A) is the same in all n amino acid units.Similarly, it is not required that R^(B) is the same in all n amino acidunits. Optionally, one or more of the side-chain functionalities maycontain a protecting group.

The amino acid units of the (oligo)peptide nucleophile may in principlebe selected from any amino acid, proteinogenic or non-proteinogenic.

In particular, the (oligo)peptide nucleophile may be represented by acompound of Formula II.

Herein, n, R^(A) and R^(B) are as defined above.

Herein P² represents an amine moiety or an OR moiety.

In case P² represents an amine moiety, the amine moiety may berepresented by the formula NR₃R₄, in which R₃ and R₄ may eachindividually represent any (substituted or unsubstituted) alkyl or(substituted or unsubstituted) aryl group. In particular, one out of R₃and R₄ is a hydrogen atom and the other a (substituted or unsubstituted)alkyl group. Good results have particularly been obtained with R₃ and R₄both being a hydrogen atom.

In case P² represents an OR moiety, R may represent a C-terminalprotective group or a cation, for instance a monovalent cation, such asa tri- or tetrasubstituted ammonium ion or an alkaline metal cation oran H. In case R is a C-terminal protective group this may in particularbe an optionally substituted alkyl group. Preferably it is a t-alkylgroup, although in principle it also may be any other protective esteras known to a man skilled in the art. The t-alkyl may in principle beany protective tertiary alkyl group. Preferably the t-alkyl is selectedfrom the group of t-butyl (2-methyl-2-propyl), t-pentyl(2-methyl-2-butyl) and t-hexyl (2,3-dimethyl-2-butyl).

In an embodiment, the (oligo)peptide nucleophile is C-terminalprotected. In another embodiment it is not C-terminal protected.

The (oligo)peptide nucleophile may be synthesized using methods known inthe art, such as solid-phase synthesis, solution phase synthesis or byfermentation using a microorganism. The N-terminal amino acid of the(oligo)peptide nucleophile and the other amino acids of the(oligo)peptide nucleophile may in principle be any amino acid,proteinogenic or non-proteinogenic. If the amino acid sequence of theN-terminal part of the (oligo)peptide nucleophile is poorly recognizedby or inaccessible to the coupling enzyme due to the amino acidpreference of the coupling enzyme or due to the secondary or tertiarystructure of the (oligo)peptide nucleophile, the primary structure(amino acid sequence) may be elongated at the N-terminus. Essentiallythe N-terminus of the (oligo)peptide nucleophile is elongated with anumber of amino acids to ensure good recognition by and accessibility tothe coupling enzyme for the enzymatic coupling reaction. The skilledperson will know how to elongate the (oligo)peptide nucleophile on thebasis of the information disclosed herein and common general knowledge.Usually the number of amino acids for elongation is in the range of1-10, although in principle it can be higher. Good result have beenobtained by elongation of the (oligo)peptide nucleophile with 3 aminoacid residues, e.g. H-Ser-Tyr-Arg.

The invention provides an enzyme having catalytic activity with respectto the formation of a peptide bond (condensation activity), whereby ithas catalytic activity in the synthesis of an (oligo)peptide with a highS/H ratio. In particular, the enzyme has ligase activity or cyclaseactivity, i.e. catalytic activity in the cyclization of an(oligo)peptide by catalyzing the formation of a peptide bond by couplingthe C-terminus and the N-terminus of an (oligo)peptide.

In particular, the invention provides an isolated enzyme (isolated fromthe organism wherein it has been expressed (typically a recombinantorganism), if it has been produced in an organism or from the reactionmedium in which it has been synthesized.

In particular, an enzyme of the invention is considered isolated for thepurpose of the invention if it has been substantially purified by anysuitable technique such as, for example, the single-step purificationmethod disclosed in Smith and Johnson, Gene 67:31-40 (1988).

An enzyme of the present invention can be provided in at leastsubstantially pure form (e.g. more than 75 wt. %, more than 80 wt. %) orin a mixture with one or more other components, e.g. in the form of astock solution, in particular in an aqueous buffer solution.

This enzyme is typically a subtilisin BPN′ variant or homologue thereof.The present disclosure provides various examples of enzymes of theinvention, which are in particular considered subtilisin BPN′ variants.As already described above, an enzyme of the invention should compriseat least:

-   -   a deletion of the amino acids corresponding to L75, N76, N77,        S78, I79, G80, V81, L82 and G83 of subtilisin BPN′ (Δ75-83; thus        in general a deletion of a corresponding Ca²⁺ binding site)    -   a cysteine or selenocysteine at a position corresponding to        position 221 in subtilisin BPN′    -   preferably an amino acid different from proline at position        corresponding to position 225 in subtilisin BPN′.

It has surprisingly been found that a mutant having both the deletioncorresponding to Δ75-83 of subtilisin BPN′ and the mutation to acysteine corresponding to position 221 in subtilisin BPN′ has sufficientstability and an S/H ratio of more than 1, which is an improved S/Hratio compared to, e.g. subtiligase. The position corresponding to S221in a subtilisin is considered to be important for stability and activityof the enzyme, and of alcalase it has been reported that a singlemutation corresponding to S221C results in a virtually inactive enzyme.In this respect, good results have been achieved with the mutation intocysteine at a position corresponding to position 221.

An enzyme of the invention may have further mutations compared tosubtilisin BPN′, provided that it has enzymatic fragment condensation orcyclisation activity in the preparation of an (oligo)peptide, inparticular one or more further mutations as described elsewhere herein.

Alternatives to subtilisin BPN′, as template enzymes from which anenzyme according to the invention, in particular a homologue of asubtilisin BPN′ variant of the invention, can be derived by mutagenesisare other subtilisins, in particular subtilisins having at least 50%homology with subtilisin BPN′.

Sequences of suitable subtilisins can be retrieved from the UNIPROTsequence database (http://www.uniprot.org/), as available on 11 Aug.2014, by BLASTing the database with subtilisin BPN′ (SEQ ID 2) as aquery. However sequence retrieval is not limited to UNIPROT nor to thedate. The skilled person in the art knows how to query alternativesequence depositories or to collect additional homologue sequences bysequencing (see for example Zooming in on metagenomics: molecularmicrodiversity of Subtilisin Carlsberg in soil., Gabor E, Niehaus F,Aehle W, Eck J. J Mol Biol. 2012 Apr. 20; 418(1-2):16-20). Inparticular, the invention further relates to variants, having at leastsaid deletions of the amino acids corresponding to L75 till andincluding G83 of subtilisin BPN′, cysteine at a position correspondingto position 221 in subtilisin BPN′ and alanine or another mutation atposition corresponding to position 225 in subtilisin BPN′ (such as amutation corresponding to P225N, 225D, P225S, P225C, P225G, P225A,P225T, P225V, P225I, P225L, P225H, P225Q of SEQUENCE ID NO: 2) of any ofthe subtilisins mentioned in FIG. 14, of which the full sequence is asavailable from said UNIPROT sequence data base and of which thealignments around positions 75-83 are shown.

Preferably, the subtilisin BPN′ variant or homologue of the inventioncomprises a mutation at the position corresponding to P225. For animprovement in S/H ratio, the mutation is usually a mutationcorresponding to P225 selected from the group of P225N, P225D, P225S,P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, P225Q, P225F andP225E. For an improvement of the S/H ratio compared to, e.g.,subtiligase a mutation is preferred corresponding to P225 selected fromthe group of P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V,P225I, P225L, P225H and P225Q. Of these, particularly good results havebeen achieved with said mutation into one of the amino acids of thegroup commonly referred to as ‘Asx’, i.e. asparagine (Asn/N) andaspartic acid (Asp/D), i.e. the mutation corresponding to P225N orP225D. Further, particularly good results have been achieved with themutation corresponding to P225S. Further, particulary good results havebeen achieved with the mutation corresponding to P225C.

Further, good results have been achieved with the mutation correspondingto P225G. Further, good results have been achieved with the mutationcorresponding to P225A. Further, good results have been achieved withthe mutation corresponding to P225T. Further, good results have beenachieved with the mutation at the position corresponding to P225 into abranched amino acid, i.e. valine (V), isoleucine (I) or Leucine (L).

Preferably, the subtilisin BPN′ variant or homologue of the inventioncomprises one or more mutations at an amino acid position correspondingto Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, 5188, Q206,N212, N218S, T254 and Q271 of SEQUENCE ID NO 2. The inventors found thatone or more of the following mutations are advantageous in thesubtilisin BPN′ variant of the invention: Q2K, S3C, P5S, S9A, I31L,K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254Aand Q271E. In particular for an improved activity, an improved stabilityor an improved S/H ratio it is preferred that a plurality of saidmutations are present in an enzyme of the invention, such as at leasttwo, at least three, more preferably four or more, more preferably fiveor more, more preferably six or more, more preferably at least eight,more preferably at least 12 of the mutations selected from the group ofQ2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P,Q206C, N212G, N218S, T254A and Q271E. The inventors consider that inparticular the presence of one or more of the mutations N218S,S3C-Q206C, G169A, T254A, A73L, M50F and Q2K are advantageous withrespect to improving enzyme stability. Further, the inventors considerthat in particular the presence of one or more of the mutations 1314E156S, G166S, G169A, is advantageous with respect to improving activityand/or S/H ratio.

Further, a subtilisin BPN′ variant or homologue according to theinvention comprising a plurality of mutations at an amino acid positioncorresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169,S188, Q206, N212, N218S, T254 and Q271 of SEQUENCE ID NO 2 is easier toproduce and purify than subtiligase.

In a preferred embodiment, the enzyme, comprises a mutation at the aminoacid position corresponding to N218, in particular N218S.

In a preferred embodiment, the enzyme, comprises a mutation at the aminoacid position corresponding to M50, in particular M50F.

In a preferred embodiment, the enzyme comprises a mutation at the aminoacid position corresponding to Q2, in particular Q2K.

In a preferred embodiment, the enzyme comprises a mutation at the aminoacid position corresponding to A73, in particular A73L.

In a preferred embodiment, the enzyme comprises a mutation at the aminoacid position corresponding to P5, in particular P5S.

In a preferred embodiment, the enzyme comprises a mutation at the aminoacid position corresponding to G166, in particular G166S.

In a preferred embodiment, the enzyme comprises a mutation at the aminoacid positions corresponding to S3 and Q206, in particular 53C-Q206C

For an improved S/H ratio, it is particularly preferred that the enzymecomprises a mutation at each of the positions corresponding to Q2, P5,M50, A73 and N218, more in particular at each of the positionscorresponding to Q2, P5, M50, A73, G166 and N218.

In particular, good results have been achieved with a subtilisin BPN′variant comprising each of the mutations corresponding to Q2K, S3C, P5S,S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G,N218S, T254A and Q271E.

Further, it has surprisingly been found that the S/H ratio is improvedin general or for certain substrates,by altering the 51′ pocket or theS4 pocket by site-specific mutations in one or more of the amino acidpositions of said pockets. It is in particular surprising that asite-specific mutation in a pocket, in particular the P1′ pocket has aneffect in another pocket, in particular the P2′ pocket. The inventorsrealized that this is also advantageous for broadening the range ofsubstrates that can advantageously be used in a method for synthesizinga peptide according to the invention. Thus this broadens the substratescope for which an enzyme of the invention offers a high S/H ratio.

The S1′ pocket is mainly formed by the amino acids M222 and Y217(Strausberg L. et al. Biochemistry, 2005, 44, 3272; Estell D. A. et al.J. Biol. Chem., 1985, 260, 6518). The three dimensional structure of theS1′ binding pocket may also be altered by more distant amino acids, forinstance N62, G100, 5125, L126, G127, P129, N155 and N218. Substitutionof one or more of these amino acids may significantly alter and improvethe S/H ratio of the subtilisin BPN′ variant or homologue, at least fora number of peptide sequences. In an advantageous embodiment, thesubstitution at an amino acid position corresponding to M222 or Y217increases the activity, S/H ratio or substrate scope for which theenzyme shows a (highly) improved S/H ratio.

Preferably, a mutation is present at the position corresponding to M222is M222G, M222P, M222N, M222E, M222Q or M222A. In a particularlypreferred embodiment, said mutation corresponds to M222P or M222G.

Preferably, a mutation at the position corresponding to Y217 is Y217L,Y217N, Y217E, Y217G, Y217S, Y217F or Y217H.

Particularly good results have been obtained with a variant having amutation selected from the group of M222G, M222P and Y217L in that theS/H ratio and/or the activity of the resulting subtilisin BPN′ variantsignificantly increases, at least for a number of peptide sequences.

The S4 binding pocket is mainly formed by the amino acids Y104, I107,L126, S101, G102, G127, and G128, but the three-dimensional structure ofthe S4 binding pocket is also determined by more distant amino acidssuch as L135 and P168 (Ruan et al. Biochemistry, 2008, 47, 6628;Rheinnecker et al. Biochemistry, 1994, 33, 221).

Preferably, the enzyme comprises a mutation at one, two or each of thepositions corresponding to Y104, I107 and L135. Particularly goodresults have been obtained with a subtilisin BPN′ variant having amutation selected from the group of Y104F, Y104S, I107V, I107A, L135N,L135S, L135D and L135A. Substitution of these amino acids cansignificantly alter and improve the S/H ratio and/or the activity of theenzyme, at least for certain substrates.

In particular, good results with respect to P4 substrate scope, enzymeactivity and S/H ratio have been obtained with a subtilisin BPN′ varianthaving a substitution in the amino acid corresponding to 1107 (I107V)and a substitution in L135 (L135S or L135N).

In a preferred embodiment, the enzyme of the invention has one or moresubstitutions in the S1′ binding pocket and one or more substitutions inthe S4 binding pocket, in particular two or more substitutions in theS1′ binding pocket and two or more substitutions in the S4 bindingpocket.

A substitution of both the amino acids corresponding to M222 and I107has been found advantageous for providing an enzyme with improvedactivity and S/H ratio compared to a variant of the invention havingonly one of said mutations. Either mutation alone was also foundbeneficial for the enzyme activity and S/H ratio. In particular, goodresults in such embodiment have been achieved by mutations I107V andM222G. Examples of other combinations of mutations of specific interestare variants with mutations L135N+M222G and variants with mutations1107V+M222P. Further, such a combination of mutations at the positionscorresponding to I107 and M222 offers improvement with respect tosubstrate scope for both the P4 and the P1′ pocket.

In a preferred embodiment, the subtilisin BPN′ variant or homologuethereof according to the invention, has a substitution in the S1′binding pocket at the position corresponding to M222 and at the positioncorresponding to Y217. The M222 mutation in this embodiment preferablyis either M222G or M222P. The Y217 mutation preferably is one selectedfrom the group of Y217F, Y217H and Y217G. Such enzymes of the inventionhave been found to have a broad substrate scope and a good S/H ratio.Particularly good results have been achieved with a subtilisin BPN′variant or homologue thereof comprising the mutations M222P and Y217H;the mutations M222P and Y217G; the mutations M222G and Y217F; or themutations M222G and Y217G. Of these, a subtilisin BPN′ variant orhomologue thereof comprising the mutations M222G and Y217F gaveparticularly good results with respect to substrate broadness and S/Hratio.

Good results have been achieved with a subtilisin BPN′ variant orhomologue thereof according to the invention having a substitution inthe S1′ binding pocket at the position corresponding to M222 and at theposition corresponding to Y217 that is free of mutations in the S4binding pocket. However, in an alternative embodiment, with which alsogood results have been achieved, it additionally has one or moremutations in the S4 binding pocket. In a specific embodiment, thissubtilisin BPN′ variant or homologue thereof has a substitution in twoor more positions of the S4 binding pocket corresponding to Y104, I107,L126, L135, 5101, G102, G127, and G128. The mutations in the S4 bindingpocket may in particular include I107V and/or either L135N or L135S.

Preferred enzymes according to the invention are in particular thesubtilisin BPN′ variant or homologues comprising any one of thesequences represented by SEQUENCE ID NO 3, 4 or 5 or homologues thereof.SEQUENCE ID NO 3 shows the preferred mutation corresponding to S221C,although in another embodiment this can be selenocysteine. The X at theposition corresponding to P225 can be P, or a different amino acidpreferably one of the preferred mutations identified elsewhere herein(N/D/S/C/G/A/T/V/I/LH/Q). SEQUENCE ID NO 4 shows preferred mutationsites compared to SEQUENCE ID NO 3. In SEQUENCE ID NO 4, each Xindependently represents any proteinogenic amino acid. In particular,any X can be the amino acid present in the wild type subtilisin BPN′ atthe position of that X or a mutation as described elsewhere in thepresent disclosure. Preferably, one or more X's represent a mutation, asdescribed elsewhere herein.

In the method of the invention the enzymatic coupling reactions andcyclisations are performed in a fluid comprising water. Preferably thereaction is performed in a buffered fluid. The water content usually is10-100 vol %, based on total liquids, preferably 20 vol. % or more,preferably 40 vol. % or more, in particular 50 vol. % or more inparticular 60 vol. % or more.

In principle, any buffer is suitable. Good buffers are known to a personskilled in the art. See for instance David Sheehan in PhysicalBiochemistry, 2^(nd) Ed. Wiley-VCH Verlag GmbH, Weinheim 2009;http://www.sigmaaldrich.comdife-science/core-bioreagents/biological-buffers/learning-center/buffer-calculator.html.

The pH of the buffer for an (oligo)peptide fragment condensation may beat least 5, in particular at least 6, preferably at least 7. A desiredmaximum pH is usually less than 11, in particular less than 10, evenmore preferably less than 9. Usually the optimal pH for the enzymaticreactions is between 7 and 9. For cyclisation reactions the optimal pHcan be different. The pH for the cyclisation reaction may be at least 3,in particular at least 4, preferably at least 5. A desired maximum pH isusually less than 11, in particular less than 10, preferably less than9. Usually the optimal pH for the enzymatic cyclisation reactions isbetween 5 and 9.

Due to the high S/H ratio, a large excess of the (oligo)peptideC-terminal ester or thioester or of the (oligo)peptide nucleophile isgenerally not needed to reach a high yield in the condensation reaction.Usually the ratio of (a) the (oligo)peptide C-terminal ester orthioester to (b) the (oligo)peptide nucleophile is between 1:5 and 5:1,preferably in the range of 1:3 to 3:1, more preferably in the range of1.0:2.5 to 2.5:1.0, in particular in the range of 1:2 to 2:1, more inparticular in the range of 1:1.5 to 1.5:1. An about stoichiometric ratiohas been found particularly effective.

In the method of the invention, it may be advantageous to add additivesto the fluid wherein the reaction is carried out to improve thesolubility of the (oligo)peptide fragments or to improve the reactionyield. Such additives may be a salt or an organic molecule, for instanceguanidinium hydrochloride, urea, sodium dodecasulphate or Tween.

The reaction may be carried out in a fully aqueous liquid or in amixture of water and a water mixable co-solvent such asN,N-dimethylformamide (DMF), N-methyl-pyrrolidinone (NMP),N,N-dimethylacetamide (DMA), dimethylsulphoxide (DMSO), acetonitrile, anether, such as tetrahydrofuran (THF), 2-methyl-tetrahydrofuran (Me-THF)or 1,2-dimethoxyethane, or a (halogenated) alcohol, such as methanol,ethanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol (TFE),1,1,1,3,3,3-hexafluoroisopropanol, or a mixture of these organicsolvents. Depending on the stability of the subtilisin BPN′ variant andthe solubility of the (oligo)peptide substrates, the amount ofco-solvent is preferably below 70 vol %, more preferably below 60 vol %,even more preferably below 50 vol %, and most preferably below 40%.

In principle the temperature during the enzymatic fragment condensationsor cyclisations is not critical, as long as a temperature is chosen atwhich the subtilisin BPN′ variant used show sufficient activity andstability. Such a temperature is usually known for the subtilisin BPN′variant to be used or can be routinely determined, making use of a knownsubstrate for the subtilisin BPN′ variant under known reactionconditions. Generally, the temperature may be at least −10° C., inparticular at least 0° C. or at least 10° C. Generally, the temperaturemay be 70° C. or less, in particular 60° C. or less or 50° C. or less.Optimal temperature conditions can easily be identified for a specificsubtilisin BPN′ variant for a specific enzymatic fragment condensationor cyclisation by a person skilled in the art through routineexperimentation based on common general knowledge and the informationdisclosed herein. In general, the temperature advantageously is in therange of 20-50° C.

The subtilisin BPN′ variants of the present invention are generallyproduced by recombinant methods, in particular by expression of asubtilisin BPN′ DNA which has been mutated such that upon expression itresults in a subtilisin BPN′ variant of the invention which isenzymatically active.

Expression of the DNA of the subtilisin BPN′ variants and homologuesthereof of the present invention is provided using available vectors andregulatory sequences. The actual selection depends in large part uponthe particular host cells which are utilized for expression. Forexample, if the subtilisin BPN′ mutant DNA is expressed in Bacillus, aBacillus promoter is generally utilized as well as a Bacillus derivedvector.

In order to produce and secrete the enzyme of the invention from a hostcell into the medium, a gene may be used which encodes a precursorpolypeptide (enzyme) containing a signal sequence and a pre-pro sequencepreceding the mature enzyme. In subtilisin BPN′, the additionalN-terminal sequence comprises 107 amino acids. Upon secretion first thesignal sequence can be removed and after secretion the pre-pro sequencecan be removed resulting in the fully active enzyme (James A. Wells,Nucleic Acids Research, Volume 11 Number 22 1983). In case of nativesubtilisin BPN′ the mature enzyme comprises 275 amino acids.Conveniently to describe the position of individual amino acids in thepolypeptide chain of subtilisin BPN′ and its homologues the so calledsubtilisin BPN′ numbering is used which runs from the N-terminus (aminoacid 1) tot the C-terminus (amino acid 275). Corresponding positions inhomologous enzymes can be determined by aligning said homologoussequences with the sequence of subtilisin BPN′.

As is known to the person skilled in the art, it is possible that the N-and/or C-termini of the mature polypeptide numbered 1-275 within SEQ IDNO: 5 or of the mature enzyme in the amino acid sequence according toSEQ ID NO: 2, 3 or 4 (as set out in amino acids 1 to 275) maybeheterogeneous, due to variations in processing during maturation. Inparticular such processing variations might occur upon overexpression ofthe enzyme. In addition, exo-protease activity might give rise toheterogeneity. The extent to which heterogeneity occurs depends also onthe host and fermentation protocols that are used. Such C-terminalprocessing artefacts might lead to shorter polypeptides or longerpolypeptides than indicated with the mature wild-type subtilisin BPN′(SEQ ID NO: 2) or with the mature enzymes according to the inventionrepresented by SEQ ID NO: 3 or 4. As a result of such processingvariations the N-terminus might also be heterogeneous. Processingvariants at the N-terminus could be due to alternative cleavage of thesignal sequence by signal peptidases.

The enzyme of the invention may be produced by recombinant technology,based on common general knowledge and the information disclosed herein.For secretion of the translated enzyme into the lumen of the endoplasmicreticulum, into the periplasmic space or into the extracellularenvironment, an appropriate secretion signal sequence may be fused tothe polynucleotide encoding the enzyme of the invention. The signals maybe endogenous to the enzyme or they may be heterologous signals.

The enzyme according to the invention may be produced in a modifiedform, such as a fusion protein, and may include not only secretionsignals but also additional heterologous functional regions. Thus, forinstance, a region of additional amino acids (a so called tag),particularly charged amino acids, may be added to the enzyme, inparticular to the C-terminus of the enzyme, to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage or to facilitate the purification. Examples ofsuitable tags are for instance described in a review by M. E. Kimple etal., in ‘Current Protocols in Protein Science 9.9.1-9.9.23, August2013’. A well known example of a useful tag is the so called His tag, anamino acid sequence having a plurality of histidine units. The inventorsfound that such a tag could be used successfully in the production andpurification of enzymes of the invention. No substantial differences infunctional enzyme properties were observed between enzymes with the Histag and enzymes without the His tag.

Further, an enzyme of the invention can be produced as an inclusion bodywith refolding in an appropriate buffer.

Enzymes of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the enzymes of the present invention may be glycosylated or may benon-glycosylated. In addition, enzymes of the invention may also includean initial modified methionine residue, in some cases as a result ofhost-mediated processes.

Polynucleotides of the invention can be incorporated into a vector,including cloning and expression vectors. A vector may be a recombinantreplicable vector. The vector may be used to replicate a polynucleotideof the invention in a compatible host cell. The vector may convenientlybe subjected to recombinant DNA procedures.

The invention also pertains to methods of growing, transforming ortransfecting such vectors in a suitable host cell, for example underconditions in which expression of an enzyme of the invention occurs. Theinvention provides a method of making enzymes of the invention byintroducing a polynucleotide of the invention into a vector, in anembodiment an expression vector, introducing the vector into acompatible host cell, and growing the host cell under conditions whichbring about replication of the vector.

The vector may be recovered from the host cell.

A vector according to the invention may be an autonomously replicatingvector, i.e. a vector which exists as an extra-chromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid.

Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

One type of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be inserted.Another type of vector is a viral vector, wherein additional DNAsegments can be inserted into the viral genome.

Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., bacterial integration vector without a suitable origin ofreplication or a non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome.

The recombinant expression vectors of the invention comprise apolynucleotide of the invention in a form suitable for expression of thepolynucleotide in a host cell, which means that the recombinantexpression vector includes one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which is operablylinked to the polynucleotide sequence to be expressed. The termregulatory sequence includes promoters, enhancers and other expressioncontrol elements (e.g., polyadenylation signal). Such regulatorysequences are described, for example, in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990).

A vector or expression construct for a given host cell may thus comprisethe following elements operably linked to each other in a consecutiveorder from the 5′-end to 3′-end relative to the coding strand of thesequence encoding an enzyme of the invention: (1) a promoter sequencecapable of directing transcription of the nucleotide sequence encodingthe enzyme in the given host cell; (2) a ribosome binding site tofacilitate the translation of the transcribed RNA (3) optionally, asignal sequence capable of directing secretion of the enzyme from thegiven host cell into a culture medium; (4) a polynucleotide sequenceaccording to the invention; and preferably also (5) a transcriptiontermination region (terminator) capable of terminating transcriptiondownstream of the nucleotide sequence encoding the enzyme.

Downstream of the nucleotide sequence according to the invention theremay be a 3′ untranslated region containing one or more transcriptiontermination sites (e.g. a terminator, herein also referred to as a stopcodon). The origin of the terminator is less critical. The terminatorcan, for example, be native to the DNA sequence encoding the enzyme.However, preferably a bacterial terminator is used in bacterial hostcells and a filamentous fungal terminator is used in filamentous fungalhost cells. More preferably, the terminator is endogenous to the hostcell (in which the nucleotide sequence encoding the enzyme is to beexpressed). In the transcribed region, a ribosome binding site fortranslation may be present. The coding portion of the mature transcriptsexpressed by the constructs will include a start codon is usually AUG(or ATG), but there are also alternative start codons, such as forexample GUG (or GTG) and UUG (or TTG), which are used in prokaryotes.Also a stop or translation termination codon is appropriately positionedat the end of the polypeptide to be translated.

Enhanced expression of the polynucleotide of the invention may also beachieved by the selection of homologous and heterologous regulatoryregions, e.g. promoter, secretion leader and/or terminator regions,which may serve to increase expression and, if desired, secretion levelsof the protein of interest from the expression host and/or to providefor the inducible control of the expression of an enzyme of theinvention.

The enzymes according to the invention can be produced in bacterialcells such as E. coli and Bacilli, insect cells (using baculovirusexpression vectors), fungal cells, yeast cells or mammalian cells.Suitable host cells are discussed herein and further in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) and in “Production of Recombinant Proteins: NovelMicrobial and Eukaryotic Expression Systems”, 2004, Wiley-Blackwell,Editor(http://eu.wiley.com/WileyCDA/Section/id-302479.html?query=Gerd+Gellissen).

Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

For most bacteria, filamentous fungi and yeasts, the vector orexpression construct is preferably integrated in the genome of the hostcell in order to obtain stable transformants. In case the expressionconstructs are integrated in the host cells genome, the constructs areeither integrated at random loci in the genome, or at predeterminedtarget loci using homologous recombination, in which case the targetloci preferably comprise a highly expressed gene.

In the invention, bacteria, in particular Bacilli, may preferably beused as host cells for the expression of an enzyme of the invention.Suitable inducible promoters useful in such host cells include promotersregulated primarily by an ancillary factor such as a repressor or anactivator. The repressors are sequence-specific DNA binding proteinsthat repress promoter activity. The transcription can be initiated fromthis promoter in the presence of an inducer that prevents binding of therepressor to the operator of the promoter. Production of secondary sigmafactors can be primarily responsible for the transcription from specificpromoters. Attenuation and antitermination also regulates transcription.

Strong constitutive promoters are well known and an appropriate one maybe selected according to the specific sequence to be controlled in thehost cell. A variety of promoters can be used that are capable ofdirecting transcription in the recombinant host cells of the invention.Preferably the promoter sequence is from a highly expressed gene. VectorDNA can be introduced into prokaryotic or eukaryotic cells via naturalcompetence, conventional transformation or transfection techniques. Asused herein, the terms “transformation” and “transfection” are intendedto refer to a variety of art-recognized techniques for introducingforeign polynucleotide (e.g., DNA) into a host cell, including calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(supra) and other laboratory manuals.

In order to identify and select cells which harbor a vector, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the polynucleotideof the invention. Preferred selectable markers include, but are notlimited to, those which confer resistance to drugs or which complement adefect in the host cell. They also include e.g. versatile marker genesthat can be used for transformation of most filamentous fungi and yeastssuch as acetamidase genes or genes providing resistance to antibioticslike G418, hygromycin, bleomycin, kanamycin, methotrexate, phleomycinorbenomyl resistance (benA). Alternatively, specific selection markerscan be used such as auxotrophic markers which require correspondingmutant host strains: e.g. D-alanine racemase (from Bacillus), URA3 (fromS. cerevisiae or analogous genes from other yeasts), pyrG or pyrA (fromA. nidulans or A. niger), argB (from A. nidulans or A. niger) or trpC.In an embodiment the selection marker is deleted from the transformedhost cell after introduction of the expression construct so as to obtaintransformed host cells capable of producing enzymes of the inventionwhich are free of selection marker genes.

Expression of proteins in prokaryotes is often carried out with vectorscontaining constitutive or inducible promoters directing the expressionof either fusion or non-fusion proteins. Fusion vectors add a number ofamino acids to a protein encoded therein, e.g. to the amino terminus ofthe recombinant protein. Such fusion vectors typically serve threepurposes: 1) to increase expression of recombinant protein; 2) toincrease the solubility of the recombinant protein; and 3) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

Vectors preferred for use in bacteria are for example disclosed inWO-A1-2004/074468, which are hereby enclosed by reference. Othersuitable vectors will be readily apparent to the skilled artisan.

Vectors of the invention may be transformed into a suitable host cell asdescribed herein to provide for expression of a polypeptide of theinvention. Thus, in a further aspect the invention provides a processfor preparing an enzyme according to the invention which comprisescultivating a host cell transformed or transfected with an expressionvector encoding the enzyme, and recovering the expressed polypeptide.

A polynucleotide according to the invention encodes, when transformedinto a proper host cell an enzyme according to the invention. Theinvention features cells, e.g., transformed host cells or recombinanthost cells comprising a polynucleotide according to the invention orcomprising a vector according to the invention. A “transformed hostcell” or “recombinant host cell” is a cell into which a polynucleotideaccording to the invention has been introduced, by means of recombinantDNA techniques.

Both prokaryotic and eukaryotic cells are included, e.g., bacteria,fungi, yeast, insect, mammalian and the like.

Suitable host cells include bacteria, including Escherichia, Anabaena,Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus,Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium),Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus,Methylobacterium, Staphylococcus Streptomyces, and Pseudomonas, In anaspect of the vector according to the invention, the host cell is abacterial cell selected from the group consisting of B. subtilis, B.puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans,Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobactersphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans,C. glutamicum, Staphylococcus carnosus, Streptomyces lividans,Sinorhizobium melioti and Rhizobium radiobacter.

In a further embodiment of the vector according to the invention thesuitable host cell is an Aspergillus, Chrysosporium, Kluyveromyces,Penicillium, Saccharomyces, or Talaromyces species.

Preferably the host cell is a Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, Escherichia coli, AspergillusNiger or Aspergillus oryzae species.

The recombinant host cell according to the invention may comprise thepolynucleotide according to the invention or the vector according to theinvention. In an embodiment of the recombinant host cell according theinvention, the recombinant host cell is capable of expressing orover-expressing the polynucleotide according to the invention or thevector according to the invention.

The method according to the invention for manufacturing thepolynucleotide according to the invention or the vector according to theinvention comprises the steps of culturing a host cell transformed withsaid polynucleotide or said vector and isolating said polynucleotide orsaid vector from said host cell.

Preferred are cells of a Bacillus strain, e.g., Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firm us, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans orStreptomyces murinus; or from a gram negative bacterium, e.g., E. colior Pseudomonas sp. (Long Liu et a., Appl Microbiol Biotechnol (2013)97:6113-6127 and Kay Terpe, Appl Microbiol Biotechnol (2006)72:211-222).

According to another aspect, the host cell is a eukaryotic host cell. Inan embodiment the eukaryotic cell is a fungal cell, i.e. a yeast cell,such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia strain. Preferably the yeast cell is aKluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowialipolytica, Pichia pastoris, or a filamentous fungal cell.

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatelyaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium,Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium andTrichoderma. In an embodiment, filamentous fungal cells are usedbelonging to a species of an Aspergillus, Chrysosporium, Penicillium,Talaromyces, Fusarium or Trichoderma genus, and preferably a species ofAspergillus niger, Aspergillus awamori, Aspergillus foetidus,Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii,Aspergillus oryzae, Chrysosporium lucknowense, Myceliophthorathermophila, Fusarium oxysporum, Trichoderma reesei or Penicilliumchrysogenum.

A host cell can be chosen which modifies and processes the encodedenzyme in a specific, desired fashion after translation. Such posttranslational modification (e.g., glycosylation) and processing (e.g.,cleavage) of protein products may facilitate optimal functioning of theprotein. Various host cells have characteristic and specific mechanismsfor post-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems familiar to thoseskilled in the art of molecular biology and/or microbiology can bechosen to ensure the desired and correct modification and processing ofthe foreign protein produced. E.g., in an embodiment a subtilisin BPN′variant or homologue thereof is initially secreted as a pre-pro-enzymeand the presence of the 77 amino acid pro sequence is important for invivo production of mature subtilisin but has to be cleaved off to obtainfull catalytic activity.

A method of producing an enzyme according to the invention typicallycomprises cultivating a recombinant host cell e.g. transformed ortransfected with an expression vector under conditions to provide forexpression of a coding sequence encoding the enzyme and recovering andpurifying the produced enzyme from the cell or culture medium.Polynucleotides of the invention can be incorporated into a recombinantreplicable vector, e. g. an expression vector or a replication vector.Transcription vectors are used to amplify their insert.

The purpose of a vector which transfers genetic information to anothercell is typically to isolate, multiply, or express the insert in thetarget cell. Vectors called expression_vectors (expression constructs)specifically are for the expression of the transgene in the target cell,and generally have a promoter sequence that drives expression of thetransgene. Simpler vectors called transcription vectors are only capableof being transcribed but not translated: they can be replicated in atarget cell but not expressed, unlike expression vectors. Transcriptionvectors are used to amplify their insert. Thus in a further embodiment,the invention provides a method of making a polynucleotide of theinvention by introducing a polynucleotide of the invention into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about thereplication of the vector. The vector may be recovered from the hostcell.

Preferably, the enzyme according to the invention is produced as asecreted protein in which case the nucleotide sequence encoding a matureform of the enzyme in the expression construct is operably linked to anucleotide sequence encoding a signal sequence. Preferably the signalsequence is native (homologous), also referred to herein as “wild type”to the nucleotide sequence encoding the enzyme. Alternatively the signalsequence is foreign (heterologous) to the nucleotide sequence encodingthe enzyme, in which case the signal sequence is preferably endogenousto the host cell in which the nucleotide sequence according to theinvention is expressed. Examples of suitable signal sequences forbacilli can be found in “van Dijl, J. M. et al. 2001. In: Sonenshein, A.L., Hoch, J. A. and Losick, R., eds. Bacillus subtilis and its closestrelatives: from genes to cells. Washington, D.C.: ASM Press, pp.337-355” and “Degering C et al., Appl Environ Microbiol. 2010 October;76(19):6370-6.”

Expression of heterologous proteins in yeast is well known. Sherman, F.,et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982)is a well recognized work describing the various methods available toexpress proteins in yeast. Vectors, strains, and protocols forexpression in, e.g. Saccharomyces and Pichia are generally known in theart and available from commercial suppliers (e.g., Invitrogen). Suitablevectors usually have expression control sequences, such as promoters,including 3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired. Morespecifically, suitable yeast signal sequences are those from yeastalfa-factor genes. Similarly, a suitable signal sequence for filamentousfungal host cells is e.g. a signal sequence from a filamentous fungalamyloglucosidase (AG) gene, e.g. the A. niger g/aA gene. This may beused in combination with the amyloglucosidase (also called (gluco)amylase) promoter itself, as well as in combination with otherpromoters. Hybrid signal sequences may also be used with the context ofthe present invention. Preferred heterologous secretion leader sequencesare those originating from the fungal amyloglucosidase (AG) gene(g/aA-both 18 and 24 amino acid versions e.g. from Aspergillus), the[alpha]-factor gene (yeasts e.g. Saccharomyces and Kluyveromyces) or the[alpha]-amylase (amyE, amyQ and amyL) and alkaline protease aprE andneutral protease genes (Bacillus).

A heterologous host cell may also be chosen wherein the enzyme of theinvention is produced in a form which is substantially free of enzymaticactivities that might interfere with the applications, e.g. free frompeptide degrading or modifying enzymes. In particular in the case ofproducing variants, the host cell should not produce any wild typeenzyme. This may be achieved by choosing a host cell which does notnormally produce such enzymes or by deliberately removing thecorresponding genes by techniques known in the art.

The invention encompasses processes for the production of the enzyme ofthe invention by means of recombinant expression of a DNA sequenceencoding the enzyme of the invention. For this purpose the DNA sequenceof the invention can be used for gene amplification and/or exchange ofexpression signals, such as promoters, secretion signal sequences, inorder to allow economic production of the enzyme in a suitablehomologous or heterologous host cell. A homologous host cell is a hostcell which is of the same species or which is a variant within the samespecies as the species from which the DNA sequence is obtained. The hostcell may over-express the enzyme, and techniques for engineeringover-expression are well known. The host may thus have two or morecopies of the encoding polynucleotide (and the vector may thus have twoor more copies accordingly). Therefore in one embodiment of theinvention the recombinant host cell according to the invention iscapable of expressing or overexpressing a polynucleotide or vectoraccording to the invention.

Another aspect of the invention is a method for producing an enzyme ofthe invention comprising (a) culturing a recombinant host cell accordingto the invention under conditions such that the enzyme of the inventionis produced ; and (b) optionally recovering the enzyme of the inventionfrom the cell culture medium. For each combination of a promoter and ahost cell, culture conditions are available which are conducive to theexpression the DNA sequence encoding the enzyme. After reaching thedesired cell density or titer of the enzyme the culture is stopped andthe enzyme is recovered. The term “culturing” includes maintainingand/or growing a living recombinant host cell of the present invention,in particular the recombinant host cell according to the invention.

In one aspect, a recombinant host cell of the invention is cultured inliquid media. In another aspect, a recombinant host cell is cultured insolid media or semi-solid media. Preferably, the recombinant host cellof the invention is cultured in liquid media comprising nutrientsessential or beneficial to the maintenance and/or growth of therecombinant host cell. The recombinant host cells may be cultured inliquid media either continuously or intermittently, by conventionalculturing methods such as standing culture, test tube culture, shakingculture, aeration spinner culture or fermentation. Preferably, therecombinant host cells are cultured in a fermentor. Fermentationprocesses of the invention include batch, fed-batch and continuousmethods of fermentation. A variety of such processes have been developedand are well known in the art.

The recombinant host cells are preferably cultured under controlled pH.In one embodiment, recombinant host cells may be cultured at a pH ofbetween 4.5 and 8.5, preferably 6.0 and 8.5, more preferably at a pH ofabout 7. The desired pH may be maintained by any method known to thoseskilled in the art.

Preferably, the recombinant host cells are further cultured undercontrolled aeration and under controlled temperatures. In oneembodiment, the controlled temperatures include temperatures between 15and 70° C., preferably the temperatures are between 20 and 55° C., morepreferably between 30 and 50° C. The appropriate conditions are usuallyselected based on the choice of the expression host and the protein tobe produced.

In a specific embodiment, the enzyme is expressed in Bacillus strainGX4935 (see examples). The strain is cultivated under aerobic conditionsin a suitable fermentation medium. A suitable medium medium may containassimilable sources of carbon and nitrogen besides inorganic saltsoptionally together with growth promoting nutrients, such as yeastextract. Fermentation is typically conducted at 35-40° C. and at a pH of6.5-7.5 and preferably kept approximately constant by automatic means.The enzyme is excreted into the medium. At the end of fermentation, ifrequired, the production host may be killed by means known by the personskilled in the art. The ensuing fermentation broth may be freed ofbacterial cells, debris therefrom together with other solids, forexample by filtration or centrifugation. The filtrate or supernatantcontaining the enzyme may be further clarified, for example byfiltration or centrifugation, and then concentrated as required, forexample by ultrafiltration or in an evaporator under reduced pressure togive a concentrate which, if desired, may be taken to dryness, forexample by lyophilization or spray-drying.

After fermentation, if necessary, the cells can be removed from thefermentation broth by means of centrifugation or filtration. Afterfermentation has stopped or after removal of the cells, the enzyme ofthe invention may then be recovered and, if desired, purified andisolated by conventional means, including, but not limited to, treatmentwith a conventional resin, treatment with a conventional adsorbent,alteration of pH, solvent extraction, dialysis, filtration,concentration, crystallization, recrystallization, pH adjustment,lyophilisation and the like. For example, the enzymes according to theinvention can be recovered and purified from recombinant cell culturesby methods known in the art (Protein Purification Protocols, Methods inMolecular Biology series by Paul Cutler, Humana Press, 2004). Usually,the compound is “isolated” when the resulting preparation issubstantially free of other components.

In an embodiment, an isolated enzyme preparation is provided having apurity of about 80% (by dry weight) of the enzyme of the invention ormore (i.e. less than about 20% of all the media, components orfermentation byproducts). In a specific embodiment, the inventionprovides the enzyme of the invention in a purity of about 90% or more,preferably in a purity of 95% or more, in particular in a purity of 98%or more. In practice, a minor amount of other components may be presentin an isolated enzyme preparation of the invention. Thus, a purifiedpreparation of the enzyme may comprise 99% or less of the enzyme, inparticular 98% or less.

Alternatively, however, the enzyme of the invention is not purified fromthe recombinant host cell or the culture. The entire culture or theculture supernatant may be used as a source of the enzyme. In a specificembodiment, the culture or the culture supernatant comprising the enzymeis used without substantial modification.

It is further noted that it is also possible to make the enzyme of theinvention, such as the subtilisin BPN′ variant, by known chemicalprotein synthesis technology, e.g. by solid phase peptide synthesis.However, expression of the subtilisin mutants in microbial host cellswill generally be preferred since this will allow for the microbial hostcell to produce the subtilisin protein in a proper conformation forenzymatic activity. However, it should be possible to convert improperlyfolded subtilisin BPN′ variants or homologues thereof into an activeconformation.

The enzymes of the invention (subtilisin BPN′ variants or homologuesthereof) may be chemically or biochemically modified, e.g.post-translationally modified. For example, they may be glycosylated orcomprise modified amino acid residues. They may also be modified by theaddition of a tag, as already mentioned above. Such modifiedpolypeptides and proteins fall within the scope of the term “enzyme” ofthe invention.

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat the same is intended only as illustrative and in nowise limitative.

EXAMPLES

Production of Enzymes (for use) According to the Invention

Mutagenesis, Cloning and Expression

The gene coding for subtilisin BS149 (Ruan et al. 2008) was obtainedfrom Philip N. Bryan (University of Maryland Biotechnology Institute,9600 Gudelsky Drive, Rockville, Md. 20850). Mutagenesis was performedusing a pUB110 based Escherichia. coli-Bacillus subtilis (E. coli-B.subtilis) shuttle vector harboring the BS149 gene using either thenative promotor or alternatively using the aprE promotor and optionallya C-terminal his-tag (pBE-S DNA, http://www.clontech.com/takara). Thegene encoding an enzyme according to the invention was constructed byintroducing the mutations S221C and P225A into the BS149 gene using thesite-directed mutagenesis method (Sambrook et al.,1989). All primerswere designed using the Agilent Primer design toolttp://www.genomics.agilent.com). The constructed sequences were verifiedby DNA sequencing before transformation to Bacillus subtilis GX4935.

In order to produce BS149-DM without a His-tag the gene coding forBS149-DM and its natural promoter sequence was cloned into the pBS42shuttle vector (DSMZ, Germany) at EcoRI/BamHI sites. The ligationmixtures were transformed into competent Escherichia coli andtransformants were plated on LB plates containing chloramphenicol (34μg/mL). The plasmid pBS42-S5 was propagated in E. coli, isolated andvalidated by sequencing. The sequence validated plasmid was used totransform B. subtilis host.

The gene coding for BS149-DM with a His-tag was cloned into a pUB-110based E. coli-B. subtilis shuttle vector (pBES) using the MluI and BamHIsite (FIG. 12). The polynucleotide sequence of a gene (BS149-DM)encoding an enzyme (polypeptide) of the invention and the encoded enzymeis shown in SEQUENCE ID NO 5. The corresponding amino acid sequence isnumbered according to the subtilisin BPN′ numbering scheme. Amino acids−107 to −1 comprise the signal sequence, the pre sequence and a prosequence which are cleaved off upon full maturation. Amino acids 1-275comprise the mature enzyme which exhibits the full catalytic activity.In order to enable a fast and efficient purification after amino acid275 a C-terminal His-tag is attached as shown in SEQUENCE ID NO 5. As aconsequence of the removal of a calcium binding site BS149-DM contains adeletion of 9 amino acids compared to subtilisin BPN′ comprising theamino acids corresponding to L75, N76, N77, S78, I79, G80, V81, L82 andG83 in subtilisin BPN′. In order to maintain the subtilisin BPN′numbering for BS149-DM the numbering jumps from 74 to 83. In the shuttlevector, the expression of the gene is under the control of aprEpromoter. The vector contained the pUB ori of replication for Bacillusand a kanamycin resistance marker. The vector also contained the ColE1ori of replication and an ampicillin resistance marker for maintenancein E. coli. The resulting plasmid pBES-BS149DMHIS was propagated in E.coli TOP10 and transformed into B. subtilis GX4935 (ΔnprEΔaprE).). UsingpBES-BS149DMHIS as the template, mutagenesis was carried out by theQuikchange method (Agilent). Alternatively other methods for sitedirected mutagenesis known in the art may be used (Sambrook etal.,1989.).

The gene of Subtiligase (Abrahmsen et al. 1991) was ordered at DNA2.0ttps://www.dna20.com/) in a DNA2.0 pJ201 cloning vector and reclonedinto E. coli-B. subtilis shuttle vector (pBS42 DSM 8748 obtained fromDSMZ; pBS42-S5). The pJ201 vector (DNA 2.0) harboring Subtiligase aswell as the pBS42 shuttle vector (DSMZ) were digested with EcoRI andBamHI (NEB). Linearized shuttle vector as well as Subtiligase insertwere isolated from gel and ligated (LigaFast, Promega). The constructwas transformed to E. coli MM294 strain (DSMZ). The plasmid pBS42-S5 waspropagated in E. coli, isolated and validated by sequencing (FIG. 13).The validated DNA was used for transformation of either B. subtilisDB104 or B. subtilis GX4935. The B. subtilis GX4935 strain has reducedextracellular proteolytic activity (Kawamura and Doi 1984; Fahnestockand Fisher 1987). The addition of the wild type subtilisin to promoteproduction of the mature form as reported by Abrahmsén et al. 1991 wasnot necessary.

Except for subtiligase and for the BS149-DM without His-tag in Example23, in all experiments, enzymes were prepared making use of a C-terminalHis-tag.

Production and Purification of Synthetic Subtilisin BPN′ Variants:

Transformants in pBS42 shuttle vector were picked and grown on LB platecontaining 10 μg/mL chloramphenicol at 37° C. for 16 h, were picked andinoculated into 5 mL of LB broth containing 10 μg/mL chloramphenicol.After 16 h of incubation at 37° C., 1% (v/v) of the cultures wereinoculated to 1 liter terrific broth (12 g/l tryptone, 24 g/l yeastextract, 0.4% (v/v) glycerol, 17 mM KH₂PO₄ and 72 mM K₂HPO₄, 50 mg/LTrp, 50 mg/L Lys, 50 mg/L Met). Cultures were grown at 37° C. withvigorous shaking and incubation was continued for 48 hours. After 48 hexpression, cells were isolated from the medium by centrifugation at6,000 g for 20 min, 4° C. Subsequently, 5 g of CaCl₂ were added to themedium and the pH was adjusted back to 7.5. The precipitate was pelletedby centrifugation at 6,000 g for 20 min, 4° C. Ammoniumsulfate was addedto the supernatant to a final concentration of 45% (w/v) to precipitatethe enzyme. The precipitated enzyme was harvested by centrifugation at8,000 g for 40 min, 4° C. The pellet was washed with 80% acetone, andresuspended in 15 mL water. The protein sample was desalted using aHiPrep 26/10 desalting column (GE healthcare) in buffer (20 mM Tricine,1 mM CaCl₂ pH 7.5). The desalted proteins were loaded on a HiTrap Q HPcolumn (GE healthcare). Flow through, which contains the enzyme, wascollected and concentrated. The purity of the protein was analyzed bySDS-PAGE and enzyme concentration was determined by measuring theabsorbtion at 280 nm (Stoscheck, C M. Quantitation of Protein. Methodsin Enzymology 182: 50-69. 1990) e.g. by NanoDrop spectrophotometer(Thermo Fisher Scientific Inc). The specific extinction coefficient canbe calculated at http://web.expasy.org/protparam/ according to GasteigerE., Hoogland C., Gattiker A., Duvaud S., Wilkins M. R., Appel R. D.,Bairoch A.; Protein Identification and Analysis Tools on the ExPASyServer; (In) John M. Walker (ed): The Proteomics Protocols Handbook,Humana Press (2005). pp. 571-607. Purity of about 90% or more wasfeasible. The obtained enzyme was provided at a concentration of about 2mg/mL in an aqueous solution in 20 mM Tricine, 1 mM CaCl₂ pH 7.5. Thisenzyme solution was used as such for the (oligo)peptide fragmentcondensations and cyclisations.

Production and Purification of Synthetic Subtilisin BPN′ Variants whichCarry a His-tag:

A single microbial colony of B. subtilis containing a plasmid with thesubtilisin variant gene of interest was inoculated in 5 mL LB withkanamycin (10 μg/mL) at 37° C. in a shaking incubator. To the 30 mLTerrific Broth supplemented with antibiotic (kanamycin 10 μg/mL) andamino acids (100 mg/L Trp, 100 mg/L Met and 100 mg/L Lys) 0.6 mL of theovernight culture was added. The cells were grown 48 h at 37° C. in ashaking incubator (200 rpm). The cells were harvested by centrifugation(15 min, 4,000 rpm, 4° C.). The medium (30 mL) was decanted andconcentrated on Amicon-centrifugal unit (15 ml, 10 kDa MW cut-off) intwo centrifugation steps (15 min, 4000 rpm, 4° C.). The concentratedmedium (0.5 ml) was then exchanged for buffer A (25 mM Tricine, pH 7.5,0.5M NaCl, 20 mM imidazole) in three washing/concentrating steps (14 mlbuffer A, 10 min, 4,000 rpm, 4° C.). For His-tag purification Talonresin (2.5 ml, Clonetech) was added to a plastic column cartridge. Theresin was washed with 5 mL MilliQ water and equilibrated with 5 mL ofbuffer A. The crude enzyme was loaded on the column and washed with 5 mLbuffer A. The enzyme was eluted with 5 mL buffer B (25 mM Tricine, pH7.5, 0.5M NaCl, 200 mM imidazole). The elute was concentrated on aAmicon-centrifugal unit (5 ml, 10 kDa MW cut-off) by centrifugation (15min, 4000 rpm, 4° C.) and the buffer was exchanged to 25 mM Tricine, pH7.5 in three washing/concentrating steps (5 ml buffer, 10 min, 4, 000rpm, 4° C.).

The purity and enzyme concentration was determined as described abovePurity was more than 90%, The obtained aqueous solution (25 mM Tricine,pH 7.5) containing about 2 mg/ml of the obtained enzyme was used as suchfor the (oligo)peptide fragment condensations and cyclisations.

REFERENCES

-   Abrahmsén, L, J Tom, J Burnier, K A Butcher, A Kossiakoff, and J A    Wells. 1991. “Engineering Subtilisin and Its Substrates for    Efficient Ligation of Peptide Bonds in Aqueous Solution.”    Biochemistry 30 (17) (April 30): 4151-9.    http://www.ncbi.nlm.nih.gov/pubmed/2021606.-   Fahnestock S R, Fisher K E: Expression of the staphylococcal protein    A gene in Bacillus subtilis by gene fusions utilizing the promoter    from a Bacillus amyloliquefaciens alpha-amylase gene. J Bacteriol.    1986 March; 165(3):796-804-   Kawamura, Fujio, and Roy H. Doi. Construction of a Bacillus subtilis    double mutant deficient in extracellular alkaline and neutral    proteases. J Bacteriol. 1984 October; 160(1): 442-4-   Ruan, Biao, Viktoriya London, Kathryn E Fisher, D Travis Gallagher,    and Philip N Bryan. Engineering substrate preference in subtilisin:    structural and kinetic analysis of a specificity mutant.    Biochemistry. 2008 Jun. 24; 47(25):6628-36.-   Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: A    Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press,    Cold Spring Harbor, N.Y.-   Wells, James A, Eugenio Ferrari, Dennis J Henner, David A Estell,    and Ellson Y Chen. Cloning, sequencing, and secretion of Bacillus    amyloliquefaciens subtilisin in Bacillus subtilis.-   Nucleic Acids Res. 1983 Nov. 25; 11(22):7911-25.

Enzymatic Fragment Condensation and Cyclisation Examples Materials andMethods

Unless stated otherwise, chemicals were obtained from commercial sourcesand used without further purification. Analytical HPLC was performed onan HP1090 Liquid Chromatograph, using a reversed-phase column(Phenomenex, C18, 5 μm particle size, 150×4.6 mm) at 40° C. UV detectionwas performed at 220 nm using a UV-VIS 204 Linear spectrometer. Thegradient program was: 0-25 min linear gradient ramp from 5% to 98%eluent B and from 25.1-30 min 5% eluent B (eluent A: 0.5 mL/L methanesulfonic acid (MSA) in H2O, eluent B 0.5 mL/L MSA in acetonitrile). Theflow was 1 mL/min from 0-25.1 min and 2 mL/min from 25.2-29.8 min, thenback to 1 mL/min until stop at 30 min. Injection volumes were 20 μL.Preparative HPLC was performed on a Varian PrepStar system using astationary-phase column (Pursuit XRs, C18, 10 μm particle size, 500×41.4mm). LC-MS was performed on an Agilent 1200 series Liquid Chromatograph,using a reversed-phase column (Phenomenex, C18, 5 μm particle size,150×4.6 mm) at 40° C. UV detection and gradient program were asdescribed for analytical HPLC. The molecular weights were determinedusing an Agilent 6130 quadrupole LC/MS system.

Protocol 1: N-Fmoc-Protected (oligo)peptide-OCam Esters were Synthesizedas Described Below:

1 gram of Rink resin (4-((2,4-dimethoxyphenyl)(Fmocamino)methyl)phenoxyalkyl linker, with a loading of 0.64 mmol/gram) waswashed with dichloromethane (DCM, 2×2 min, 10 mL) and1-methyl-2-pyrrolidone (NMP, 2×2 min, 10 mL) and Fmoc-deprotected usingpiperidine/NMP (1/4, v/v, 2×8 min, 10 mL). After washing with NMP (2×2min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), iodoaceticacid (4 equiv.) was coupled to the resin using DCC (4 equiv.) and HOAt(4 equiv.) in DCM (45 min, 10 mL). After washing with NMP (2×2 min, 10mL), DCM (2×2 min, 10 mL) and THF (2×2 min, 10 mL), the resin was loadedwith an Fmoc-protected amino acid using 4 equiv. Fmoc-Xxx-OH and 10equiv. DiPEA in DMF/THF (1/1, v/v, 10 mL) at 50° C. for 20 h. Here andin other parts of this disclosure ‘Xxx’ stands for one amino acid(variable as indicated in the Figures belonging to the examples below).

After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP(2×2 min, 10 mL), standard SPPS protocols were followed to elongate thepeptide (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage fromthe resin and side-chain deprotection was performed using a mixture oftrifluoroacetic acid (TFA), triisopropylsilane (TIS) and water(95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptide wasprecipitated using methyl tert-butyl ether (MTBE)/n-heptanes (1/1, v/v,50 mL). The precipitated peptide was collected by centrifugation andwashed twice with MTBE/n-heptanes (1/1, v/v, 50 mL) followed bylyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 2: N-Fmoc-Protected (oligo)peptide-OCam-Xxx-NH₂ Esters wereSynthesized as Described Below:

1 gram of Rink resin was washed with DCM (2×2 min, 10 mL) and NMP (2×2min, 10 mL) and Fmoc-deprotected using piperidine/NMP (1/4, v/v, 2×8min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10mL) and NMP (2×2 min, 10 mL), Fmoc-Xxx-OH (4 equiv.) was coupled to theresin using HBTU (4 equiv.), HOBt (4 equiv.) and DiPEA (8 equiv.) in NMP(45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min,10 mL) and NMP (2×2 min, 10 mL), the resin was Fmoc-deprotected usingpiperidine/NMP (1/4, v/v, 2×8 min, 10 mL). After washing with NMP (2×2min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), iodoaceticacid (4 equiv.) was coupled using DCC (4 equiv.) and HOAt (4 equiv.) inDCM (45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2min, 10 mL) and THF (2×2 min, 10 mL), an Fmoc-protected amino acid wascoupled using 4 equiv. Fmoc-Xxx-OH and 10 equiv. DiPEA in DMF/THF (1/1,v/v, 10 mL) at 50° C. for 20h. After washing with DMF (2×2 min, 10 mL),DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), standard SPPS protocolswere followed to elongate the peptide (Weng C. Chan and Peter White, OUPOxford, 2000). Cleavage from the resin and side-chain deprotection wasperformed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL)for 120 min. The crude peptide was precipitated using MTBE/n-heptanes(1/1, v/v, 50 mL). The precipitated peptide was collected bycentrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL)followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 3: N-Fmoc-Protected (oligo)peptide-OCam-Xxx-OH Esters wereSynthesized as Described Below:

1 gram of Trityl resin (2-chloro-chlorotrityl linker, with a loading of1.0 mmol/gram) was washed with DCM (2×2 min, 10 mL) and Fmoc-Xxx-OH (2equiv.) was coupled to the resin using DiPEA (5 equiv.) in DCM (30 min,10 mL). After washing with DMF (2×2 min, 10 mL), the unreactedchlorotrityl groups were capped using DCM/MeOH/DiPEA (80/15/5, v/v/v,2×10 min, 10 mL). The resin was washed with NMP (2×2 min, 10 mL), DCM(2×2 min, 10 mL) and NMP (2×2 min, 10 mL) and Fmoc-deprotected usingpiperidine/NMP (1/4, v/v, 2×8 min, 10 mL). After washing with NMP (2×2min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), iodoaceticacid (4 equiv.) was coupled using DCC (4 equiv.) and HOAt (4 equiv.) inDCM (45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2min, 10 mL) and THF (2×2 min, 10 mL), an Fmoc-protected amino acid wascoupled using 4 equiv. Fmoc-Xxx-OH and 10 equiv. DiPEA in DMF/THF (1/1,v/v, 10 mL) at 50° C. for 20h. After washing with DMF (2×2 min, 10 mL),DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), standard SPPS protocolswere followed to elongate the peptide (Weng C. Chan and Peter White, OUPOxford, 2000). Cleavage from the resin and side-chain deprotection wasperformed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL)for 120 min. The crude peptide was precipitated using MTBE/n-heptanes(1/1, v/v, 50 mL). The precipitated peptide was collected bycentrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL)followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 4: (oligo)peptide C-Terminal Amide Nucleophiles wereSynthesized as Described Below:

1 gram of Rink resin(4-((2,4-dimethoxyphenyl)(Fmoc-amino)methyl)-phenoxyalkyl linker, withaloading of 0.64 mmol/gram) was washed with DCM (2×2 min, 10 mL) and NMP(2×2 min, 10 mL) and Fmoc-deprotected using piperidine/NMP (1/4, v/v,2×8 min, 10 mL). Standard SPPS protocols were followed to elongate thepeptide (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage fromthe resin and side-chain deprotection was performed using a mixture ofTFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptidewas precipitated using MTBE/n-heptanes (1/1, v/v, 50 mL). Theprecipitated peptide was collected by centrifugation and washed twicewith MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization fromacetonitrile/water (1/1, v/v, 50 mL).

Protocol 5: N-Acetyl-Protected (oligo)peptide Activated Esters wereSynthesized as Described Below:

After SPPS of the desired sequence according to one of the protocols1-3, the resin bound peptide was Fmoc-deprotected using piperidine/NMP(1/4, v/v, 2×8 min, 10 mL). The resin was washed with NMP (2×2 min, 10mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL) and the peptideN-terminal amine function was acetylated using a mixture of Ac₂O (10 vol%), DiPEA (5 vol %), HOBt (0.2 wt %) in NMP (2×10 min, 10 mL). The resinwas washed with NMP (3×2 min, 10 mL) and DCM (3×2 min, 10 mL). Cleavagefrom the resin and side-chain deprotection was performed using a mixtureof TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crudepeptide was precipitated using MTBE/n-heptanes (1/1, v/v, 50 mL). Theprecipitated peptide was collected by centrifugation and washed twicewith MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization fromacetonitrile/water (1/1, v/v, 50 mL).

Protocol 6: (oligo)peptide C-Terminal Acids were Synthesized asDescribed Below:

1 gram of Trityl resin (2-chloro-chlorotrityl linker, with a loading of1.0 mmol/gram) was washed with DCM (2×2 min, 10 mL) and Fmoc-Xxx-OH (2equiv.) was coupled to the resin using DiPEA (5 equiv.) in DCM (30 min,10 mL). After washing with DMF (2×2 min, 10 mL), the unreactedchlorotrityl groups were capped using DCM/MeOH/DiPEA (80/15/5, v/v/v,2×10 min, 10 mL). The resin was washed with NMP (2×2 min, 10 mL), DCM(2×2 min, 10 mL) and NMP (2×2 min, 10 mL) and standard SPPS protocolswere followed to elongate the peptide (Weng C. Chan and Peter White, OUPOxford, 2000). Cleavage from the resin and side-chain deprotection wasperformed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL)for 120 min. The crude peptide was precipitated using MTBE/n-heptanes(1/1, v/v, 50 mL). The precipitated peptide was collected bycentrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL)followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 7: Synthesis Partially Protected (oligo)peptide Fragments

During SPPS of the peptide sequence according to one of the protocols1-6, at the desired position a differently (TFA stable) protected aminoacid was coupled such as Fmoc-Asp(OcHex)-OH, Fmoc-Glu(OBn)-OH orFmoc-Lys(Alloc)-OH. Cleavage from the resin and side-chain deprotection,except for the TFA stable cHex, Bn or Alloc group which remainedunaffected, was performed using a mixture of TFA/TIS/water (95/2.5/2.5,v/v/v, 15 mL) for 120 min. The crude peptide was precipitated usingMTBE/n-heptanes (1/1, v/v, 50 mL). The precipitated peptide wascollected by centrifugation and washed twice with MTBE/n-heptanes (1/1,v/v, 50 mL) followed by lyophilization from acetonitrile/water (1/1,v/v, 50 mL).

COUPLING EXAMPLES

Note: The enzyme denoted as BS149-DM (SEQUENCE ID NO:5) contains adeletion of amino acids 75-83 and mutations Q2K, S3C, P5S, S9A, I31L,K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L,N218S, S221C, P225A, T254A and Q271E compared to SEQUENCE ID NO:2. Onthe basis of the present disclosure, common general knowledge andoptionally a limited amount of route testing, the skilled person in theart may revert one or more of mutations Q2K, S3C, PSS, S9A, I31L, K43N,M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S,T254A and Q271E or make different substitutions at one or more of thepositions Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, 5188,Q206, N212, N218S, T254, Q271 while still having significantly improvedproperties compared to Subtiligase (see for instance example 24).

The enzyme denoted as Subtiligase contains the mutations S221C and P225Acompared to SEQUENCE ID NO:2.

The enzymes of the invention used in the Examples 1-23 have all themutations of BS149-DM, plus optional additional mutations as mentionedin the Examples.

As indicated below, enzymes with further mutations were made using thetechnology described above.

Example 1 Enzymatic Oligopeptide Fragment Coupling Using DifferentBS149-DM Mutants

To test the activity and S/H ratio of the different mutants, thefollowing standard reaction was performed. 800 μL of phosphate buffer(100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminalamide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water)and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5μg enzyme was added and the reaction mixture was shaken (150 rpm) atroom temperature. After 30 min a 500 μL aliquot of the reaction mixturewas withdrawn and quenched with 500 μL MSA/water (1/99, v/v) andanalyzed by LC-MS. The product, hydrolysed pentapeptide C-terminalCam-ester and remaining pentapeptide C-terminal Cam-ester peaks wereintegrated.

The activity of the different BS149-DM mutants is defined as the totalof the amount of product and the amount of hydrolysed pentapeptideC-terminal Cam-ester divided by the total of the amount of product,hydrolysed pentapeptide C-terminal Cam-ester and remaining Cam-ester,within the specified reaction time. The most active mutant was set to100% (see FIG. 1A). The S/H ratio of the different BS149-DM mutants isdefined as the amount of product divided by the amount of hydrolysedpentapeptide C-terminal Cam-ester, within the specified time (see FIG.1B).

Activity and S/H ratio in other examples were determined in the sameway, unless specified otherwise.

Conclusions: clearly, BS149-DM has a twice higher activity and animproved S/H ratio (1.8 versus 0.9) as compared to subtiligase. The M222position proved very important for the S/H ratio of the enzyme.Especially good results were obtained with the M222G and M222P mutantsof BS149-DM. All BS149-DM variants containing a P4 pocket mutation(positions Y104, I107 and L135) have a comparable S/H ratio to BS149-DM.However, for certain mutations, the activity of the BS149-DM variantswas drastically improved. Particularly good results were obtained withthe mutations Y104S, I107V, L135D, L135N and L135S. When combining P4pocket mutations, BS149-DM variants with even higher activity wereobtained, i.e. I107V+L135S and 1107V +L135N. When P4 pocket mutationswere combined with P1′ pocket mutations, a very active BS149-DM variantwith an increased S/H ratio as compared to BS149-DM was obtained, e.g.I107V+M222G.

Example 2 Enzymatic Oligopeptide Fragment Coupling Using DifferentBS149-DM+M222P+L217 Mutants

To test the activity and S/H ratio of the different mutants, the samereaction as described in Example 1 was performed. The activity of thedifferent BS149-DM+M222P+L217 mutants is defined as the total of theamount of product and the amount of hydrolysed oligopeptide C-terminalCam-ester divided by the total of the amount of product, hydrolysedoligopeptide C-terminal Cam-ester and remaining Cam-ester. The mostactive mutant was set to 100% (see FIG. 2A). The S/H ratio of thedifferent BS149-DM+M222P+L217 mutants is defined as the amount ofproduct divided by the amount of hydrolysed C-terminal Cam-ester (seeFIG. 2B).

Conclusions: clearly, all BS149-DM+M222P+L217 mutants have an improvedS/H and similar or improved activity as compared to subtiligase and someof them have an increased activity compared to BS149-DM+M222P.Particularly good results were obtained with the mutations L217N, L217T,L217E, L2171, L217V and L217A. The L217 position proved not only veryimportant for activity and S/H ratio but is even more important for thesubstrates scope, as described in Example 5.

Example 3 Mapping the P4 Pocket Substrate Specificity of DifferentBS149-DM Mutants Containing a P4 Pocket Mutation (Positions Y104, I107and L135)

To determine the P4 pocket substrate specificity of the differentmutants, the following standard reaction was performed. 800 μL ofphosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μLtripeptide C-terminal amide stock solution (0.01 mmolH-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 200 μL pentapeptideC-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Xxx-Ser-Lys-Leu-OCam.TFA in 1.2 mL water+1 mL Acn). Couplingswith all these peptide esters were performed, differing in the aminoacid at this position, as indicated in FIGS. 3A-3C.

To this mixture 5.5 μg enzyme was added and the reaction mixture wasshaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot ofthe reaction mixture was withdrawn and quenched with 500 μL MSA/water(1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptideC-terminal Cam-ester and remaining pentapeptide C-terminal Cam-esterpeaks were integrated. The activity is defined as the amount of productdivided by the total of the amount of product, hydrolysed pentapeptideC-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester,within the specified reaction time. The most active substrate was set to100%, see FIGS. 3A-C.

Conclusions: as evident from FIGS. 3A-C, the P4 substrate scope of theBS149-DM mutants with a P4 mutation (on positions Y104, I107 and/orL135) clearly differs from that of BS149-DM which may be advantageousfor various particular peptide sequences. Several mutants show a muchbroader P4 substrate scope than BS149-DM. This is in particular the casewith mutations I107V, L135D, L135N and L135S.

Example 4 Mapping the P1′ and P2′ Pocket Substrate Specificity ofDifferent BS149-DM+M222 Mutants

To determine the P1′ and P2′ pocket substrate specificity of thedifferent mutants, the following two standard reactions were performed.800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of100 μL tripeptide C-terminal amide stock solution (0.01 mmolH-Xxx-Leu-Arg-NH₂.2TFA for P1′ and H-Ala-Xxx-Arg-NH₂.2TFA for P2′ in 300μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution(0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To thismixture 5.5 μg enzyme was added and the reaction mixture was shaken (150rpm) at room temperature. After 30 min a 550 μL aliquot of the reactionmixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) andanalyzed by LC-MS. The product, hydrolysed pentapeptide C-terminalCam-ester and remaining pentapeptide C-terminal Cam-ester peaks wereintegrated. The activity is defined as the amount of product divided bythe total of the amount of product, hydrolysed pentapeptide C-terminalCam-ester and remaining pentapeptide C-terminal Cam-ester, within thespecified reaction time. The most active substrate was set to 100%, TheP1′ and P2′ pocket substrate specificities of different BS149-DM+M222mutants are shown in FIGS. 4 A-D. The coupling with tryptophan in P1′position was not determined due to overlap in the LC-MS peaks.

Conclusions: as evident from FIGS. 4A-D, the P1′ and P2′ substratescopes of the BS149-DM mutants with a P1′ mutation on position M222clearly differs from that of BS149-DM which may be advantageous forvarious particular peptide sequences. Several mutants show a muchbroader P1′ and P2′ substrate scope than BS149-DM. This is in particularthe case with mutations M222G and M222P.

Example 5 Mapping the P1′ and P2′ Pocket Substrate Specificity ofDifferent BS149-DM+M222P+L217 Mutants

To determine the P1′ and P2′ pocket substrate specificity of thedifferent mutants, the same reactions and analyses were performed asdescribed in Example 4. The P1′ and P2′ pocket substrate specificitiesof different BS149-DM+M222P +L217 mutants are shown in FIGS. 5A-L.

Conclusions: as evident from FIGS. 5A-L, the P1′ and P2′ substratescopes of the BS149-DM+M222P mutants with a P1′ mutation on positionL217 clearly differs from that of BS149-DM+M222P which may beadvantageous for various particular peptide sequences. Several mutantsshow a much broader P1′ and P2′ substrate scope than BS149-DM. This isin particular the case with mutations L217G and L217H. Several mutantsshow a drastically improved activity for certain particular substrates.For instance improved activity for Phe in the P1′ pocket for mutationsBS149-DM+M222P+L217N, E, G, Y, F or II. The mutant BS149-DM+M222P+L217Halso shows a much increased activity for Asn in the P1′ pocket. Themutants BS149-DM+M222P+L217E and A have an improved activity for Leu,Ile and Val in the P1′ pocket. The mutants BS149-DM+M222P+L217T and Shave an improved activity for Asp in the P1′ pocket.

Example 6 Mapping the P1′ Pocket Substrate Specificity of DifferentBS149-DM+M222G+L217 Mutants

To determine the P1′ pocket substrate specificity of the differentmutants, the same reactions and analyses were performed as described inExample 4. The P1′ pocket substrate specificities of differentBS149-DM+M222G+L217 mutants are shown in FIGS. 6A-F.

Conclusions: as evident from FIGS. 6A-F, the P1′ substrate scopes of theBS149-DM+M222G mutants with a P1′ mutation on position L217 clearlydiffers from that of BS149-DM+M222G which may be advantageous forvarious particular peptide sequences. Several mutants show a muchbroader P1′ substrate scope than BS149-DM. This is in particular thecase with mutations L217G and L217F. Several mutants show a drasticallyimproved activity for certain particular substrates. For instanceimproved activity for Phe in the P1′ pocket for mutationsBS149-DM+M222G+L217N, E, G, Y, F, I or II. The mutantBS149-DM+M222G+L217F also shows a much increased activity for Asn in theP1′ pocket. The mutants BS149-DM+M222G+L217F, G, A and Y have animproved activity for Leu, Ile and Val in the P1′ pocket. The mutantsBS149-DM+M222G+L217R, T and S have an improved activity for Asp in theP1′ pocket.

Example 7 Mapping the P1′, P2′ and P4 Pocket Substrate Specificity ofBS149-DM+M222G+1107V Mutant

To determine the P1′ and P2′ pocket substrate specificity ofBS149-DM+M222G+I107V, the same reactions and analyses were performed asdescribed in Example 4. The P1′ and P2′ pocket substrate specificitiesof the BS149-DM+M222G+I107V mutant are shown in FIGS. 67A and B,respectively. To determine the P4 pocket substrate specificity ofBS149-DM+M222G+1107V, the same reactions and analyses were performed asdescribed in Example 3. The P4 pocket substrate specificity of theBS149-DM+M222G+I107V mutant is shown in FIG. 7C.

Conclusions: as evident from FIGS. 7A-C, the P1′ and P2′ substrate scopeas well as the P4 substrate scope of the BS149-DM+M222G+1107V mutant arebroader as compared to BS149-DM. Clearly, the advantageous mutations forthe P1′ and P2′ pockets (i.e. M222G) and for the P4 pocket (i.e. 1107V)can be successfully combined since the substrate broadness is comparableto the BS149-DM+M222G mutant but the S/H ratio is significantly higher(see Example 1).

Example 8 Enzymatic Coupling Reactions using Different N-acetylProtected Oligopeptide C-Terminal Cam-ester Acyl Donors

Peptide ligation reactions were performed at 25° C. in 100 mM Tricinebuffer (pH 8.0), containing 15 μM BS149-DM, 10 mM peptide C-terminalCam-ester (Ac-Asp-Leu-Ser-Lys-Gln-OCam.TFA,Ac-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA,Ac-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA orAc-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA) and 15 mMdipeptide C-terminal amide (H-Ala-Phe-NH₂). After 180 min the reactionmixtures were analyzed by LC-MS. The product, hydrolysed C-terminalCam-ester and remaining Cam-ester peaks were integrated. The S/H ratioof the different reactions is defined as the amount of product dividedby the amount of hydrolysed C-terminal Cam-ester, within the specifiedreaction time.

TABLE 1 Coupling of different acyl donors with H-Ala-Phe-NH2Peptide C-terminal Peptide mine S/H Cam-ester nucleophile ratioAc-Asp-Leu-Ser-Lys-Gln-Ocam H-Ala-Phe-NH₂ 12Ac-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Ocam H-Ala-Phe-NH₂ 59Ac-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys- H-Ala-Phe-NH₂ 41 Gln-OCamAc-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu- H-Ala-Phe-NH₂ 80 Ser-Lys-Gln-OCam

Conclusions: different lengths of oligopeptide acyl donors can be used.The S/H ratio increases with the length of the oligopeptide acyl donor.

Example 9 Enzymatic Coupling Reactions using Different OligopeptideC-Terminal Amide Nucleophiles

Peptide ligation reactions were performed at 25° C. in 100 mM Tricinebuffer (pH 8.0), containing 15 μM BS149-DM, 1 mM pentapeptide C-terminalCam-ester (Ac-Phe-Ile-Glu-Trp-Leu-OCam) and 3 mM peptide aminenucleophile (H-Ala-Phe-NH₂, H-Ala-Phe-Ala-NH₂ or H-Ala-Phe-Ala-Tyr-NH₂).After 60 min the reaction mixtures were analyzed by LC-MS. The product,hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptideC-terminal Cam-ester peaks were integrated. The S/H ratio of thedifferent reactions is defined as the amount of product divided by theamount of hydrolysed pentapeptide C-terminal Cam-ester, within thespecified reaction time.

TABLE 2 Coupling of different oligopeptide nucleophiles with Ac-Phe-Ile-Glu-Trp-Leu-OCam Peptide C-terminal Peptide amine S/H Cam-esternucleophile ratio Ac-Phe-Ile-Glu-Trp-Leu-OCam H-Ala-Phe-NH₂ 1.5Ac-Phe-Ile-Glu-Trp-Leu-OCam H-Ala-Phe-Ala-NH₂ 1.7Ac-Phe-Ile-Glu-Trp-Leu-OCam H-Ala-Phe-Ala-TyrNH₂ 1.9

Conclusions: different lengths of oligopeptide nucleophiles can be used.The S/H ratio increases with the length of the oligopeptide nucleophile.

Example 10 Effect of the pH on the S/H Ratio of BS149-DM+M222G Mutant

To examine the effect of pH on the S/H ratio of the BS149-DM+M222Gmutant, the following standard reaction was performed. 800 μL ofphosphate buffer (1M, pH 7.0-8.8), or tricine buffer (1M, pH 7.9-8.9) orcarbonate buffer (1M, pH 9.2-10.6) was added to a mixture of 100 μLtripeptide C-terminal amide stock solution (0.01 mmolH-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptideC-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water). To this mixture 5.5 μgenzyme was added and the reaction mixture was shaken (150 rpm) at roomtemperature. After 30 min a 550 μL aliquot of the reaction mixture waswithdrawn and quenched with 50 μL MSA and analyzed by LC-MS. Theproduct, hydrolysed pentapeptide C-terminal Cam-ester and remainingpentapeptide C-terminal Cam-ester peaks were integrated and the S/Hratio is defined as the amount of product divided by the amount ofhydrolysed pentapeptide C-terminal Cam-ester, within the specifiedreaction time, see FIG. 8.

Conclusions: the S/H ratio of BS149-DM+M222G is dependent on the pH andthere is a clear optimum between pH 8 and pH 9, but lower or higher pHcan also be used depending on the solubility and stability properties ofthe oligopeptides.

Example 11 Effect of the Concentration of Acyl Donors and Nucleophileson the S/H Ratio of BS149-DM+M222G

To examine the effect of substrate concentration on the S/H ratio ofmutant BS149-DM+M222G, the following reactions were performed. A stocksolution of tripeptide C-terminal amide (12.9 mg H-Glu-Leu-Arg-NH₂.2TFAor 11.7 mg H-Ala-Leu-Arg-NH₂.2TFA) and C-terminal pentapeptide Cam-ester(4.2 mg Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA) in 150 μL water was prepared.The mixtures were brought to neutral pH with 5.1 μL NaOH (32wt % inwater). To prepare reaction mixtures with different concentration ofsubstrates, 10 μL of one of the above stock solutions was diluted with10, 20, 50, 100, 200, 500, 1000, 2000, 5000 and 10000 μL phosphatebuffer (1M, pH 8.5). To these reaction mixtures 11 μg of BS149-DM+M222Gwas added and the reaction mixture was shaken (150 rpm) at roomtemperature. After 30 min a 550 μL aliquot of the reaction mixture waswithdrawn and quenched with 50 μL MSA and analyzed by LC-MS. Theproduct, hydrolysed pentapeptide C-terminal Cam-ester and remainingpentapeptide C-terminal Cam-ester peaks were integrated and the S/Hratio is defined as the amount of product divided by the amount ofhydrolysed pentapeptide C-terminal Cam-ester, within the specifiedreaction time, see FIGS. 9A and B.

Conclusions: the S/H ratio is dependent on the substrate concentrations.There is an optimal substrate concentration for each individualsubstrate depending on the affinity of the nucleophile for the enzymeand on the solubility and stability properties of the oligopeptides.

Example 12 Effect of Dosing of the Acyl Donor on the S/H Ratio usingBS149-DM+M222G

To examine the effect of dosing of the acyl donor on the S/H ratio ofmutant BS149-DM+M222G, the following two reactions were performed. Intwo-fold, 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100μL tripeptide C-terminal amide stock solution (0.01 mmolH-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 5.5 μg BS149-DM+M222G. Toone of these mixtures, 100 μL pentapeptide C-terminal Cam-ester stocksolution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water) wasadded and the reaction mixture was shaken (150 rpm) at room temperature.To the other mixture 100 μL pentapeptide C-terminal Cam-ester stocksolution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water) wasdosed in portions of 3.5 μL every minute while shaking the reactionmixture (150 rpm) at room temperature. After 30 min a 550 μL aliquot ofboth reaction mixtures was withdrawn and quenched with 50 μL MSA andanalyzed by LC-MS. For both reactions the conversion of the pentapeptideC-terminal Cam-ester starting material was 100%. The product andhydrolysed pentapeptide C-terminal Cam-ester peaks were integrated andthe S/H ratio is defined as the amount of product divided by the amountof hydrolysed pentapeptide C-terminal Cam-ester, within the specifiedreaction time. The S/H ratio for the reaction where all acyl donor wasadded at t=0 was 2.45 and the S/H ratio for the reaction where the acyldonor was dosed every minute was 2.73.

Conclusions: by dosing the oligopeptide C-terminal Cam-ester in time theS/H ratio can be improved.

Example 13 Cyclization of Oligopeptide C-Terminal Cam-Esters usingDifferent Enzymes

The following experiments were performed to determine the S/H ratio ofSubtiligase, BS149-DM and BS149-DM+M222G for the cyclization of anoligopeptide C-terminal Cam-ester.

800 μL of phosphate buffer (100 mM, pH 8.0) was added to a 100 μL stocksolution of an oligopeptide C-terminal Cam-ester with an N-terminal freeamine (0.01 mmolH-Ala-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-Tyr-Gln-Ser-Tyr-OCam.2TFA in 1 mLwater) containing 5 mg/mL dithiotreitol. To this mixture 5.5 μg enzymewas added and the reaction mixtures were shaken (150 rpm) at roomtemperature. After 30 min a 550 μL aliquot of the reaction mixtures waswithdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed byLC-MS. The product, hydrolysed C-terminal Cam-esterand remainingCam-ester starting material peaks were integrated and the S/H ratio ofthe different enzymes is defined as the amount of product divided by theamount of hydrolysed C-terminal Cam-ester, within the specified reactiontime, see FIG. 10.

Conclusions: evidently, also for peptide cyclisation, BS149-DM has animproved S/H ratio as compared to Subtiligase. The BS-149-DM+M222Gmutant has an even higher S/H ratio.

Example 14 Effect of the pH on the S/H Ratio During Cyclisation of anOligopeptide C-Terminal Cam-Ester using BS149-DM+M222G

To determine the effect of the pH on the S/H ratio of BS149-DM+M222Gduring the cyclisation of an oligopeptide C-terminal Cam-ester, thefollowing standard reactions were performed. 800 μL of phosphate buffer(1M, pH 5, 6, 7, 8 and 9) was added to 100 μL stock solution of atridecapeptide C-terminal Cam-ester with an N-terminal free amine (0.01mmol H-Ala-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-Tyr-Gln-Ser-Tyr-OCam.2TFA in1 mL water) containing 5 mg/mL dithiotreitol. To this mixture 5.5 μgBS149-DM+M222G was added and the reaction mixture was shaken (150 rpm)at room temperature. After 30 min a 550 μL aliquot of the reactionmixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS.The product, hydrolysed C-terminal Cam-ester and remaining C-terminalCam-ester starting material peaks were integrated and the S/H ratio isdefined as the amount of product divided by the amount of hydrolysedC-terminal Cam-ester, within the specified reaction time, see FIG. 11.

Conclusions: the S/H ratio of BS149-DM+M222G used for enzymaticoligopeptide cyclisation is dependent on the pH, albeit to a lesserextent than for enzymatic oligopeptide fragment condensation.

Example 15 Fragment Condensation with Oligopeptides over 10 Amino AcidsLong

To examine whether enzymatic fragment condensation with longeroligopeptides in aqueous solution is feasible, the following standardreaction was performed. 800 μL of phosphate buffer (1M, pH 8.0) wasadded to a mixture of 100 μL decapeptide C-terminal amide stock solution(0.01 mmol H-Ala-Leu-Met-Lys-Tyr-Asn-Ser-Thr-Glu-Val-NH₂.2TFA in 300 μLwater) and 200 μL tridecapeptide C-terminal Cam-ester stock solution(0.01 mmolFmoc-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.2TFA in1.2 mL water +1 mL DMF). To this mixture 5.5 μg BS149-DM+M222G was addedand the reaction mixture was shaken (150 rpm) at room temperature. After30 min a 550 μL aliquot of the reaction mixture was withdrawn andquenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysedC-terminal Cam-ester and remaining C-terminal Cam-ester peaks wereintegrated. The amount of product(Fmoc-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Ala-Leu-Met-Lys-Tyr-Asn-Ser-Thr-Glu-Val-NH₂),within the specified reaction time, was 68 area %.

Conclusion: Fragment condensations with longer peptides arewell-feasible.

Example 16 Fragment Condensation using Oligopeptides without N- orC-Terminal Protecting Group

To examine whether enzymatic fragment condensation without N- orC-terminal protecting group is feasible without significant side-productformation, the following standard reaction was performed. 800 μL ofphosphate buffer (1M, pH 8.0) was added to a mixture of 100 μLtripeptide C-terminal carboxylic acid stock solution (0.01 mmolH-Ala-Leu-Arg-OH.2TFA in 300 μL water) and 100 μL N-terminal free aminepentapeptide C-terminal Cam-ester stock solution (0.01 mmolH-His-Ala-Glu-Gly-Thr-OCam.TFA in 1.2 mL water). To this mixture 5.5 μgof BS149-DM+M222G was added and the reaction mixture was shaken (150rpm) at room temperature. After 30 min a 550 μL aliquot of the reactionmixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS.The product, hydrolysed pentapeptide C-terminal Cam-ester and remainingpentapeptide C-terminal Cam-ester peaks were integrated. The amount ofproduct (H-His-Ala-Glu-Gly-Thr-Ala-Leu-Arg-OH), within the specifiedreaction time,was 74 area %. No side-products were observed indicatingthat no side-reactions had occurred at the C-terminal carboxylic acidfunction of H-Ala-Leu-Arg-OH.2TFA nor at the N-terminal amine functionof H-His-Ala-Glu-Gly-Thr-OCam.

Conclusion: some oligopeptide sequences can be successfullyenzymatically ligated without using N- or C-terminal protecting groups.

Example 17 Fragment Condensations using Oligopeptide C-TerminalCam-Xxx-NH₂ or Cam-Xxx-OH Esters

To examine whether enzymatic fragment condensations with Cam-Xxx-NH₂ orCam-Xxx-OH esters are feasible, the following standard reactions wereperformed. 800 μL of phosphate buffer (1M, pH 8.0) was added to amixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmolH-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptideC-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH.TFA,Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-NH₂.TFA,Ac-Asp-Phe-Ser-Lys-Leu-OCam-Lys-NH₂.2TFA orAc-Asp-Phe-Ser-Lys-Leu-OCam-Glu-NH2.TFA in 1.2 mL water). To each ofthese 4 mixtures 5.5 μg of BS149-DM+M222G was added and the reactionmixtures were shaken (150 rpm) at room temperature. After 30 min a 550μL aliquot of the reaction mixtures was withdrawn and quenched with 50μL MSA and analyzed by LC-MS. The product, hydrolysed pentapeptideC-terminal Cam-ester and remaining tetrapeptide C-terminal Cam-esterpeaks were integrated. The amount of product(Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH₂), within the specified reactiontime, was 86 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH, 83 area %for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-NH₂, 78 area % forAc-Asp-Phe-Ser-Lys-Leu-OCam-Lys-NH₂ and 83 area % forAc-Asp-Phe-Ser-Lys-Leu-OCam-Glu-NH₂.

Conclusions: this example shows that Cam-Xxx-NH₂ and Cam-Xxx-OH esterscan be used successfully for enzymatic oligopeptide fragmentcondensation.

Example 18 Fragment Condensation using a C-Terminal OligopeptideThioester and BS149DM+I107V+M222G

To examine whether enzymatic oligopeptide fragment condensations usingC-terminal thioesters are feasible, the following standard reaction wasperformed. 1 mL of Tricine buffer (100 mM, pH 7.5), containing 2.5 mMpentapeptide C-terminal thio-ester (Suc-Ala-Ala-Pro-Phe-SBzl), 25 mMdipeptide C-terminal amide (H-Gly-Phe-NH₂), and 5 μgBS149-DM+L107V+M222G, was shaken (150 rpm) at 25° C. After 30 min a 550μL aliquot of the reaction mixture was withdrawn and quenched with 50 μLMSA and analyzed by LC-MS. The product, hydrolysed tetrapeptideC-terminal thio-ester and remaining tetrapeptide C-terminal thio-esterpeaks were integrated. The amount of product(Suc-Ala-Ala-Pro-Phe-Gly-Phe-NH₂), within the specified reaction time,was 85 area %.

Conclusions: this example shows that oligopeptide C-terminal thioesterscan be used successfully for enzymatic oligopeptide fragmentcondensation.

Example 19 Fragment Condensation Using a C-Terminal Oligopeptide AlkylEster and BS149-DM+I107V+M222G

To examine whether enzymatic oligopeptide fragment condensations usingC-terminal alkyl esters are feasible the following standard reaction wasperformed. 1 mL of Tricine buffer (100 mM, pH 7.5), containing 2.5 mMpentapeptide C-terminal alkyl ester (Ac-Asp-Phe-Ser-Lys-Leu-OTFE(TFE=2,2,2-trifluoroethyl)), 25 mM tripeptide C-terminal amide(H-Ala-Leu-Arg-NH₂), and 5 μg BS149-DM+I107V+M222G, was shaken (150 rpm)at 25° C. After 30 min a 550 μL aliquot of the reaction mixture waswithdrawn and quenched with 50 μL. MSA and analyzed by LC-MS. Theproduct, hydrolysed pentapeptide C-terminal alkyl-ester and remainingpentapeptide alkyl-ester peaks were integrated. The amount of product(Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH₂), within the specified reactiontime, was 55 area %.

Conclusions: this example shows that oligopeptide C-terminal alkylesters can be used successfully for enzymatic oligopeptide fragmentcondensation.

Example 20 Enzymatic Oligopeptide Fragment Condensation Using PartialSide-Chain Protection

To demonstrate that partial P1′ side-chain protection can be beneficial,the following reaction was performed. 800 μL of phosphate buffer (100mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amidestock solution (0.01 mmol H-Asp-Leu-Arg-NH₂.2TFA or 0.01 mmolH-Asp(OcHex)-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptideC-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5μg BS149-DM+M222G was added and the reaction mixture was shaken (150rpm) at room temperature. After 30 min a 500 μL aliquot of the reactionmixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) andanalyzed by LC-MS. The product, hydrolysed pentapeptide C-terminalCam-ester and remaining peptapeptide Cam-ester peaks were integrated.The amount of product using the unprotected substrate(H-Asp-Leu-Arg-NH₂), within the specified reaction time, was 18 area %,the amount of product using the partially side-chain protected substrate(H-Asp(OcHex)-Leu-Arg-NH₂), within the specified reaction time, was 73area %.

To demonstrate that partial P2′ side-chain protection can be beneficial,the following reaction was performed. 800 μL of phosphate buffer (100mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amidestock solution (0.01 mmol H-Asp-Glu(OBn)-Arg-NH₂.2TFA or 0.01 mmolH-Asp-Glu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptideC-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5μg BS149-DM+M222G was added and the reaction mixture was shaken (150rpm) at room temperature. After 30 min a 500 μL aliquot of the reactionmixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) andanalyzed by LC-MS. The product, hydrolysed pentapeptide C-terminalCam-ester and remaining peptapeptide Cam-ester peaks were integrated.The amount of product using the unprotected substrate(H-Asp-Glu-Arg-NH₂), within the specified reaction time, was 15 area %,the amount of product using the partially side-chain protected substrate(H-Asp-Glu(OBn)-Arg-NH₂), within the specified reaction time, was 58area %.

To demonstrate that partial P1 side-chain protection can be beneficial,the following reaction was performed. 800 μL of phosphate buffer (100mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amidestock solution (0.01 mmol H-Asp-Leu-Arg-NH₂.2TFA) and 100 μLpentapeptide C-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Phe-Ser-Leu-Lys-OCam.TFA or 0.01 mmolAc-Asp-Phe-Ser-Leu-Lys(Alloc)-OCam in 1200 μL water). To this mixture5.5 μg BS149-DM+M222G was added and the reaction mixture was shaken (150rpm) at room temperature. After 30 min a 500 μL aliquot of the reactionmixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) andanalyzed by LC-MS. The product, hydrolysed pentapeptide C-terminalCam-ester and remaining peptapeptide Cam-ester peaks were integrated.The amount of product using the unprotected substrate(Ac-Asp-Phe-Ser-Leu-Lys-OCam.TFA), within the specified reaction time,was 5 area %, the amount of product using the partially side-chainprotected substrate (Ac-Asp-Phe-Ser-Leu-Lys(Alloc)-OCam) was 84 area %.

To demonstrate that partial P4 side-chain protection can be beneficial,the following reaction was performed. 800 μL of phosphate buffer (100mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amidestock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA) and 100 μLtetrapeptide C-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Ser-Lys-Leu-OCam.TFA or 0.01 mmolAc-Asp(OBn)-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5μg BS149-DM+M222G was added and the reaction mixture was shaken (150rpm) at room temperature. After 30 min a 500 μL aliquot of the reactionmixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) andanalyzed by LC-MS. The product, hydrolysed tetrapeptide C-terminalCam-ester and remaining tetrapeptide Cam-ester peaks were integrated.The amount of product using the unprotected substrate (mmolAc-Asp-Ser-Lys-Leu-OCam.TFA), within the specified reaction time, was 32area %, the amount of product using the partially side-chain protectedsubstrate (Ac-Asp(OBn)-Ser-Lys-Leu-OCam.TFA) was 78 area %.

Conclusions: this example shows that partial side-chain protection canimprove the yield and/or reaction rate of enzymatic oligopeptidefragment condensations

Example 21 Thermostability of BS149-DM

The fluorescence-based thermal stability assay was used to determine theapparent melting temperature of BS149-DM and subtiligase. A sample of 20μL of protein solution in buffer (20 mM Tricine buffer, pH 7.5) andmetal ions (10 mM) or EDTA (10 mM) was mixed with 5 μL of 100 timesdiluted Sypro Orange (Molecular Probes, Life Technologies, USA) dye in athin wall 96-well PCR plate. The plate was sealed with Optical-QualitySealing Tape and heated in an CFX 96 Real Time PCR System (BioRad,Hercules, Calif., USA) from 20 to 99° C. at a heating rate of 1.75°C./min. Fluorescence changes were monitored with a charge-coupled device(CCD) camera. The wavelengths for excitation and emission were 490 and575 nm, respectively. The thermostability of the purified BS149-DM wasdetermined as described above. The thermostability was also determinedafter the addition of different metal ions and chelating agents, seeTable 3 below. An apparent transition temperature (Tm) of 66° C. wasobserved, indicating that the enzyme BS149-DM well preserves thethermostability from BS149. In contrast, the Tm value of Subtiligase wasdetermined to be 59° C.

TABLE 3 Effect of metal ions (10 mM) and the chelating agent EDTA (10mM) on the thermostability of BS149-DM. T_(m) (° C.) Control 66 Ca²⁺65.5 Mg²⁺ 65 Mn²⁺ 64.5 Ni²⁺ 62 EDTA 66.5

Conclusions: clearly, BS149-DM has an improved thermostability ascompared to Subtiligase. The enzyme BS149-DM is also resistant to metalions and chelating agents, since in their presence the Tm value remainsvirtually unaffected.

Example 22 Effect of Organic Solvents and Different Additives onBS149-DM Activity

Peptide ligation reactions were performed at 25° C. in 100 mM Tricinebuffer (pH 8.0), containing 15 μM BS149-DM, 1 mM pentapeptide C-terminalCam-ester (Ac-Phe-Ile-Glu-Trp-Leu-OCam) and 3 mM dipeptide C-terminalamide (H-Ala-Phe-NH₂). Different amounts of metal ions (10 mM), EDTA (10mM) or organic solvent were added and after 60 min the reaction mixtureswere analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminalCam-ester and remaining peptapeptide Cam-ester peaks were integrated.The activity of BS149-DM is defined as the total of the amount ofproduct and the amount of hydrolysed pentapeptide C-terminal Cam-esterdivided by the total of the amount of product, hydrolysed pentapeptideC-terminal Cam-ester and remaining Cam-ester, within the specifiedreaction time. The most reaction with the highest activity was set to100%, see Tables 4-8.

TABLE 4 Effect of metal ions (10 mM) and the chelating agent EDTA (10mM) on the activity of BS149-DM. Activity (%) No additive 87 Ca²⁺ 67Mg²⁺ 87 Mn²⁺ 100 Ni²⁺ 73 EDTA 87

TABLE 5 Effect of THF on the activity of BS149-DM. Activity (%) Noadditive 100 10 vol % THF 60 20 vol % THF 30 30 vol % THF 10 40 vol %THF 4

TABLE 6 Effect of DMF on the activity of BS149-DM. Activity (%) Noadditive 100 10 vol % DMF 64 20 vol % DMF 36 30 vol % DMF 32 40 vol %DMF 18 50 vol % DMF 14

TABLE 7 Effect of DMSO on the activity of BS149-DM. Activity (%) Noadditive 100 10 vol % DMSO 87 20 vol % DMSO 73 30 vol % DMSO 76 40 vol %DMSO 62 50 vol % DMSO 35

TABLE 8 Effect of GndCl on the activity of BS149-DM. Activity (%) Noadditive 92 0.66M GndCl 100 1.32M GndCl 90 2.00M GndCl 81 2.64M GndCl 753.33M GndCl 67 4.00M GndCl 43

Example 23 Enzymatic Oligopeptide Fragment Coupling using BS149-DM withand without His-tag

To test the activity and S/H ratio of the different enzymes, thefollowing two standard reactions were performed. 800 μL of phosphatebuffer (100 mM, pH 8.0) was added to a mixture of 100 μL pentripeptideC-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution(0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To thismixture was added 5.5 μg BS149-DM either with or without His-tag wasadded and the reaction mixtures were shaken (150 rpm) at roomtemperature. After 30 min a 500 μL aliquot of the reaction mixtures waswithdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed byLC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester andremaining pentapeptide C-terminal Cam-ester peaks were integrated.

The S/H ratio of BS149-DM with His-tag respectively without His-tag isdefined as the amount of product (synthesized oligopeptide) divided bythe amount of hydrolysed pentapeptide C-terminal Cam-ester, within thespecified time. The S/H ratio for BS149 with His-tag was 1.91 and forBS149 without His-tag 1.98.

The activity of BS149-DM with and without His-tag is defined as thetotal of the amount of product and the amount of hydrolysed pentapeptideC-terminal Cam-ester divided by the total of the amount of product,hydrolysed pentapeptide C-terminal Cam-ester and remaining Cam-ester,within the specified reaction time. The activity of BS149-DM withHis-tag was 97.3% and for BS149-DM without His-tag 98.6%.

Conclusions: the presence or absence of the His-tag has no significanteffect on the S/H ratio and the activity.

Example 24 S/H Ratio of Enzymes Corresponding to SEQ ID NO 3 withDifferent Mutations

To test the activity and S/H ratio of the different mutants, thefollowing standard reaction was performed. 800 μL of phosphate buffer(100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminalamide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water)and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5μg enzyme was added and the reaction mixture was shaken (150 rpm) atroom temperature. After 30 min a 500 μL aliquot of the reaction mixturewas withdrawn and quenched with 500 μL MSA/water (1/99, v/v) andanalyzed by LC-MS. The product, hydrolysed pentapeptide C-terminalCam-ester and remaining pentapeptide C-terminal Cam-ester peaks wereintegrated.

The S/H ratio of the different mutants is defined as the amount ofproduct divided by the amount of hydrolysed pentapeptide C-terminalCam-ester, within the specified time (see Table 9).

TABLE 9 S/H ratio of enzymes corresponding to SEQ ID NO 3 with differentmutations Amino acid at position S/H X225 of SEQ ID NO 3 Additivemutations ratio P (proline, as in wild-type 1.97 subtilisin BPN′) A 2.03G 1.76 A N218S 2.40 A N218S, M50F 2.55 A N218S, M50F, S3C-Q206C, Q2K,2.40 A73L, P5S A N218S, M50F, S3C-Q206C, Q2K, 2.09 A73L, P5S, Y217L

Conclusions: clearly, several enzymes corresponding to SEQ ID NO 3 (X=A)with the S221C mutation have a twofold increased S/H ratio compared tosubtiligase (S/H subtiligase=0.9, see example 1). The S/H ratio remainsunaffected with X=P, G or A.

Example 25 S/H Ratio of BS149-DM+M222P+L217H+X225 Mutants

To test the activity and S/H ratio of the different mutants, thefollowing standard reaction was performed. 800 μL of phosphate buffer(100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminalamide stock solution (0.01 mmol H-Ser-Leu-Arg-NH₂.2TFA in 300 μL water)and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmolAc-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5μg enzyme was added and the reaction mixture was shaken (150 rpm) atroom temperature. After 30 min a 500 μL aliquot of the reaction mixturewas withdrawn and quenched with 500 μL MSA/water (1/99, v/v) andanalyzed by LC-MS. The product, hydrolysed pentapeptide C-terminalCam-ester and remaining pentapeptide C-terminal Cam-ester peaks wereintegrated.

The S/H ratio of the different mutants is defined as the amount ofproduct divided by the amount of hydrolysed pentapeptide C-terminalCam-ester, within the specified time (see Table 10).

TABLE 10 S/H ratio of BS149-DM + M222P + L217H + X225 mutants Mutant S/Hratio BS149-DM + M222P + L217H + X225N 7.33 BS149-DM + M222P + L217H +X225D 6.69 BS149-DM + M222P + L217H + X225S 6.07 BS149-DM + M222P +L217H + X225C 5.25 BS149-DM + M222P + L217H + X225G 4.63 BS149-DM +M222P + L217H + X225A 4.47 BS149-DM + M222P + L217H + X225T 4.28BS149-DM + M222P + L217H + X225V 4.26 BS149-DM + M222P + L217H + X225I4.00 BS149-DM + M222P + L217H + X225L 3.55 BS149-DM + M222P + L217H +X225H 1.84 BS149-DM + M222P + L217H + X225Q 1.45 BS149-DM + M222P +L217H + X225F 0.71 BS149-DM + M222P + L217H + X225E 0.36 BS149-DM +M222P + L217H + X225P 0.17 BS149-DM + M222P + L217H + X225K 0.07BS149-DM + M222P + L217H + X225Y 0.03 BS149-DM + M222P + L217H + X225M0.03 BS149-DM + M222P + L217H + X225R 0.02 BS149-DM + M222P + L217H +X225W 0.01

Conclusions: clearly, the mutations at the X225 position have a largeeffect on the S/H ratio. Many mutations have a superb effect such aswith X=N, D, S, C, G and A. Several further enzymes have an over threefold increased S/H ratio as compared to subtiligase (S/Hsubtiligase=0.9, see example 1) such as with X=L, I, V and T. Also,mutations of X225 into H, Q, and—to a lesser extent—F and E showed animprovement over the wild-type enzyme with X225 being P.

Example 26 Coupling of a Pentapeptide Selectively to the N-Terminus ofthe A-Chain of Human Insulin

5 mg of human insulin (CAS #11061-68-0) and 2.5 mg ofAc-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH.TFA were dissolved in 200 μL DMF.Subsequently, 200 μL of phosphate buffer (1 M, pH 8.0) and 200 μL H₂Ocontaining 20 μg of the BS149-DM+M222G mutant were added and thereaction mixture was shaken (150 rpm) at room temperature. After 60 mina 100 μL aliquot of the reaction mixture was withdrawn and quenched with500 μL MSA/water (1/99, v/v) and analyzed by LC-MS, showing that 92% ofthe insulin starting material was converted to a single product, i.e.Ac-Asp-Phe-Ser-Lys-Leu- coupled to the N-terminus of the insulinA-chain.

Example 27 Coupling of a Pentapeptide to the N-Terminus of the A- andB-Chain of Human Insulin

5 mg of human insulin (CAS #11061-68-0) and 5 mg ofAc-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH.TFA were dissolved in 200 μL DMF.Subsequently, 200 μL of phosphate buffer (1 M, pH 8.0) and 200 μL H₂Ocontaining 55 μg of BS149-DM+M222G+L217F mutant were added and thereaction mixture was shaken (150 rpm) at room temperature. After 60 mina 100 μL aliquot of the reaction mixture was withdrawn and quenched with500 μL MSA/water (1/99, v/v) and analyzed by LC-MS, showing that theinsulin starting material was completely consumed and converted to threeproduct peaks, i.e. 1) Ac-Asp-Phe-Ser-Lys-Leu- coupled to the N-terminusof the Insulin A-chain (22 area %), 2) Ac-Asp-Phe-Ser-Lys-Leu- coupledto the N-terminus of the insulin B-chain (3 area %) and 3)Ac-Asp-Phe-Ser-Lys-Leu- coupled to the N-terminus of both the Insulin A-and B-chain (75 area %).

Sequences

SEQ ID NO 1: wild type gene encoding for subtilisin BPN′amino acids-107 to 275 ENA|K02496|K02496.1 B. Subtilisin BPN′Bacillus amyloliquefaciensGTGAGAGGCAAAAAAGTATGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGAT GGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATA TATTGTCGGGTTTAAACAGACAATGAGCACGATGAGCGCCGCTAAGAAGAAAGATGTCATT TCTGAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACAT TAAACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAAGA TCACGTAGCACATGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAATTAAAGCCCCT GCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTA TCGATTCTTCTCATCCTGATTTAAAGGTAGCAGGCGGAGCCAGCATGGTTCCTTCTGAAACA AATCCTTTCCAAGACAACAACTCTCACGGAACTCACGTTGCCGGCACAGTTGCGGCTCTTA ATAACTCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTTTACGCTGTAAAAGTTCT CGGTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAATCGAGTGGGCGATCGC AAACAATATGGACGTTATTAACATGAGCCTCGGCGGACCTTCTGGTTCTGCTGCTTTAAAAG CGGCAGTTGATAAAGCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCAGCCGGTAACGAAG GCACTTCCGGCAGCTCAAGCACAGTGGGCTACCCTGGTAAATACCCTTCTGTCATTGCAGTA GGCGCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTCAAGCGTAGGACCTGAGCTTGAT GTCATGGCACCTGGCGTATCTATCCAAAGCACGCTTCCTGGAAACAAATACGGGGCGTACA ACGGTACGTCAATGGCATCTCCGCACGTTGCCGGAGCGGCTGCTTTGATTCTTTCTAAGCAC CCGAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGT GATTCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGTAASEQ ID NO 2: wild type subtilisin BPN′(mature) >SUBT_BACAM Subtilisin BPN′Bacillus amyloliquefaciens mature 1 to 275AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ SEQ ID NO 3: subtilisin BPN′variant with deletion of Ca²⁺ binding loopand S221C and preferably P225 mutation (denoted as P225X)AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTCMASXHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ SEQ ID NO 4: subtilisin BPN′variant with preferred mutation positions  compared to SEQ ID NO 3AXXVXYGVXQIKAPALHSQGYTGSNVKVAVXDSGIDSSHPDLXVAGGASXVPSETNPFQDNNSHGTHVAGTVXAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNXGTSGSSSTVXYPXKYPSVIAVGAVDSSNQRAXFSSVGPELDVMAPGVSIXSTLPGXKYGAXXGTCMASXHVAGAAALILSKHPNWTNTQVRSSLENTXTKLGDSFYYGKGLINVXAAAQSEQ ID NO 5: The segment of E. coli/B. subtilis shuttle vector pBES:Pt1149DM Hiscontaining the B. subtilis-derived subtilisin (aprE) promoter region (bp 1-197, Takara), the BPN′ signal sequence (bp 198-287), the BPN′prodomain (bp 288-518), the mature B5149-DM, 6xHistag, stop codon. From nucleotide 1590 onwards the sequence follows pBES from Takara.    1ACTAGTGTTC TTTTCTGTAT GAAAATAGTT ATTTCGAGTC TCTACGGAAA TAGCGAGAGA   61TGATATACCT AAATAGAGAT AAAATCATCT CAAAAAAATG GGTCTACTAA AATATTATTC  121CATCTATTAC AATAAATTCA CAGAATAGTC TTTTAAGTAA GTCTACTCTG AACTTAAGCA  181AAAGGAGAGG GACGCGT GTG AGA GGC AAA AAA GTA TGG ATC AGT TTG CTG TTT    RBS      MluI  Val Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe                  -107     -105                -100  234GCT TTA GCG TTA ATC TTT ACG ATG GCG TTC GGC AGC ACA TCC TCT GCCAla Leu Ala Leu Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala-95                -90                  -85                 -80  282CAG GCG GCA GGG AAA TCA AAC GGG GAA AAG AAA TAT ATT GTC GGG TTTGln Ala Ala Gly Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe                -75                 -70                 -65  330AAA CAG ACA ATG AGC ACG ATG AGC GCC GCT AAG AAG AAA GAT GTC ATTLys Gln Thr Met Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile            -60                 -55                -50  378TCT GAA AAA GGC GGG AAA GTG CAA AAG CAA TTC AAA TAT GTA GAC GCASer Glu Lys Gly Gly Lys Val Gln Lys Gln Phe Lys Tyr Val Asp Ala        -45                 -40                 -35  426GCT TCA GCT ACA TTA AAC GAA AAA GCT GTA AAA GAA TTG AAA AAA GACAla Ser Ala Thr Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp    -30                 -25                 -20  474CCG AGC GTC GCT TAC GTT GAA GAA GAT CAC GTA GCA CAC GCG ATG GCGPro Ser Val Ala Tyr Val Glu Glu Asp His Val Ala His Ala Met Ala-15                 -10                 -5                   1  522AAG TGC GTG TCT TAC GGC GTA GCG CAA ATT AAA GCC CCT GCT CTG CACLys Cys Val Ser Tyr Gly Val Ala Gln Ile Lys Ala Pro Ala Leu His             5                   10                 15  570TCT CAA GGC TAC ACT GGA TCA AAT GTT AAA GTA GCG GTT CTT GAC AGCSer Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Leu Asp Ser         20                  25                  30  618GGT ATC GAT TCT TCT CAT CCT GAT TTA AAC GTA GCA GGC GGA GCC AGCGly Ile Asp Ser Ser His Pro Asp Leu Asn Val Ala Gly Gly Ala Ser     35                  40                  45  666TTC GTT CCT TCT GAA ACA AAT CCT TTC CAA GAC AAC AAC TCT CAC GGAPhe Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly50                   55                  60                  65  714ACT CAC GTT GCC GGC ACA GTT TTG GCT GTT GCG CCA AGC GCA TCA CTTThr His Val Ala Gly Thr Val Leu Ala Val Ala Pro Ser Ala Ser Leu                 70              74* 84  85                  90  762TAC GCT GTA AAA GTT CTC GGT GCT GAC GGT TCC GGC CAA TAC AGC TGGTyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp                 95                 100                 105  810ATC ATT AAC GGA ATC GAG TGG GCG ATC GCA AAC AAT ATG GAC GTT ATTIle Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn Asn Met Asp Val Ile            110                 115                 120  858AAC ATG AGC CTC GGC GGA CCT TCT GGT TCT GCT GCT TTA AAA GCG GCAAsn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala        125                 130                 135  906GTT GAT AAA GCC GTT GCA TCC GGC GTC GTA GTC GTT GCG GCA GCC GGTVal Asp Lys Ala Val Ala Ser Gly Val Val Val Val Ala Ala Ala Gly    140                 145                 150  954AAC TCT GGC ACT TCC GGC AGC TCA AGC ACA GTG AGC TAC CCT GCT AAAAsn Ser Gly Thr Ser Gly Ser Ser Ser Thr Val Ser Tyr Pro Ala Lys155                 160                 165                 170 1002TAC CCT TCT GTC ATT GCA GTA GGC GCT GTT GAC AGC AGC AAC CAA AGATyr Pro Ser Val Ile Ala Val Gly Ala Val Asp Ser Ser Asn Gln Arg                175                 180                 185 1050GCA CCG TTC TCA AGC GTA GGA CCT GAG CTT GAT GTC ATG GCA CCT GGCAla Pro Phe Ser Ser Val Gly Pro Glu Leu Asp Val Met Ala Pro Gly            190                 195                 200 1098GTA TCT ATC TGT AGC ACG CTT CCT GGA GGC AAA TAC GGG GCG CTT TCTVal Ser Ile Cys Ser Thr Leu Pro Gly Gly Lys Tyr Gly Ala Leu Ser        205                 210                 215 1146GGT ACG TGC ATG GCA TCT GCG CAC GTT GCC GGA GCG GCT GCT TTG ATTGly Thr Cys Met Ala Ser Ala His Val Ala Gly Ala Ala Ala Leu Ile    220                 225                 230 1194CTT TCT AAG CAC CCG AAC TGG ACA AAC ACT CAA GTC CGC AGC AGT TTALeu Ser Lys His Pro Asn Trp Thr Asn Thr Gln Val Arg Ser Ser Leu235                 240                 245                 250 1242GAA AAC ACC GCT ACA AAA CTT GGT GAT TCT TTC TAC TAT GGA AAA GGGGlu Asn Thr Ala Thr Lys Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly                255                 260                 265 1290CTG ATC AAC GTA GAA GCG GCA GCT CAG CAC CAC CAC CAC CAC CAC TAALeu Ile Asn Val Glu Ala Ala Ala Gln His His His His His His ---            270                 275                 280 1338AACATAAAAA ACCGGCCTTG GCCCCGCCGG TTTTTTATTA TTTTTCTTCC TCCGCATGTT 1398CAATCCGCTC CATAATCGAC GGATGGCTCC CTCTGAAAAT TTTAACGAGA AACGGCGGGT 1458TGACCCGGCT CAGTCCCGTA ACGGCCAAGT CCTGAAACGT CTCAATCGCC GCTTCCCGGT 1518TTCCGGTCAG CTCAATGCCG TAACGGTCGG CGGCGTTTTC CTGATACCGG GAGACGGCAT 1578TCGTAATCGG ATGGATCC                  BamHI *Deletion with respect toBPN′ of amino acid 72-80 (Val-Ala-Ala-Leu-Asn-Asn-Ser-Ile-Gly); GTT GCGGCT CTT AAT AAC TCA ATC GGT.

What is claimed is:
 1. A method for enzymatically synthesizing an(oligo)peptide, comprising: coupling (a) an (oligo)peptide C-terminalester or thioester and (b) an (oligo)peptide nucleophile having anN-terminally unprotected amine, wherein the coupling is carried out in afluid comprising water, and wherein the coupling is catalyzed by asubtilisin BPN′ variant or a homologue thereof, said subtilisin BPN′variant or a homologue thereof comprising mutations as compared tosubtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequencethereof, said mutations comprising: a deletion of the amino acidscorresponding to positions 75-83; and a mutation at the amino acidposition corresponding to 5221, the mutation corresponding to S221C orS221selenocysteine; and a mutation at the amino acid positioncorresponding to P225; wherein the amino acid positions are definedaccording to the sequence of subtilisin BPN′ represented by SEQUENCE IDNO:
 2. 2-12. (cancelled)
 13. A method for enzymatically synthesizing acyclic (oligo)peptide of at least 12 amino acids, comprising: subjectingan (oligo)peptide C-terminal ester or thioester having an N-terminallyunprotected amine to a cyclisation step wherein said cyclization iscarried out in a fluid comprising water, and wherein the cyclization iscatalyzed by a subtilisin BPN′ variant or a homologue thereof, saidsubtilisin BPN′ variant or a homologue thereof comprising mutations ascompared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or ahomologue sequence thereof, said mutations comprising: a deletion of theamino acids corresponding to positions 75-83; and a mutation at theamino acid position corresponding to 5221, the mutation being S221C orS221selenocysteine; and wherein the amino acid positions are definedaccording to the sequence of subtilisin BPN′ represented by SEQUENCE IDNO:
 2. 14. The method of claim 1, wherein the mutation at the amino acidposition corresponding to S221 is S221C.
 15. The method of claim 1,wherein the subtilisin BPN′ variant or homologue thereof comprises amutation at the amino acid position corresponding to P225, and whereinsaid mutation corresponds to P225N, P225D, P225S, P225C, P225G, P225T,P225V, P2251, P225L, P225H or P225Q. 16-23. (canceled)
 24. The method ofclaim 1 wherein the subtilisin BPN′ variant or homologue thereof,comprises one or more mutations, and wherein said mutations at an aminoacid position corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156,G166, G169, S188, Q206, N212, N218, T254 or Q271 of SEQUENCE ID NO 2.25. The method of claim 24, wherein said one or more mutationscorrespond to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S,G169A, S188P, Q206C, N212G, N218S, T254A or Q271E.
 26. The method ofclaim 25, wherein the subtilisin BPN′ variant or homologue thereof,comprises at least four, of said mutations, and wherein said mutationscorrespond to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S,G169A, S188P, Q206C, N212G, N218S, T254A or Q271E.
 27. The method ofclaim 26, wherein the subtilisin BPN′ variant or homologue thereof,comprises the mutations corresponding to Q2K, S3C, P5S, S9A, I31L, K43N,M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A orQ271E.
 28. The method of claim 1, wherein the subtilisin BPN′ variant orhomologue thereof, comprises one or more mutations selected from thegroup of mutations at an amino acid position corresponding to N62, G100,S125, L126, G127, P129, N155, Y217, N218 or M222 of SEQUENCE ID NO 2.29. The method of claim 28, wherein the subtilisin BPN′ variant orhomologue thereof, contains a mutation at the position corresponding toM222 of SEQUENCE ID NO
 2. 30. The method of claim 29, wherein saidmutation at the position corresponding to M222 is M222G, M222P, M222N,M222E, M222Q or M222A.
 31. The method of claim 28, wherein thesubtilisin BPN′ variant or homologue thereof, comprises a mutation atthe amino acid position corresponding to Y217 of SEQUENCE ID NO
 2. 32.The method of claim 31, wherein the mutation at Y217 is Y217L, Y217N,Y217E, Y217G, Y217F, Y217S, Y217A or Y217H.
 33. The method of claim 1,wherein the subtilisin BPN′ variant or homologue thereof, comprises atleast one mutation, and wherein said mutation is at an amino acidposition corresponding to Y104, I107, L126, S101, G102, G127, G128,L135, or P168 of SEQUENCE ID NO
 2. 34. The method of claim 33, whereinsaid mutation is at an amino acid corresponding to Y104, I107 and L135.35. The method of claim 1, wherein the (oligo)peptide C-terminal esteris defined by the formula peptide-(C═O)O—CX₂—C(═O)N—R₁R₂, each Xindependently representing a hydrogen atom or an alkyl group; and R₁representing a hydrogen atom or an alkyl group and R₂ representing ahydrogen atom or an alkyl group or an amino acid or a peptide residuewith a C-terminal carboxyamide or carboxylic acid functionality,optionally protected on the side-chain functionality of the amino acidor on one or more of the side-chain functionalities of the amino acids.36-43. (canceled)
 44. An enzyme, comprising: mutations compared tosubtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequencethereof, said mutations comprising: a deletion of the amino acidscorresponding to positions 75-83; a mutation at the amino acid positioncorresponding to S221, the mutation being a mutation corresponding toS221C or S221selenocysteine; and a mutation at the amino acid positioncorresponding to P225; wherein the amino acid positions are definedaccording to the sequence of subtilisin BPN′ represented by SEQUENCE IDNO: 2; and wherein the enzyme is a subtilisin BPN′ variant or homologuethereof.
 45. The enzyme of claim 44, wherein the mutation at the aminoacid position corresponding to S221 is a mutation corresponding toS221C. 46-53. (canceled)
 54. The enzyme of claim 44, comprising one ormore mutations, wherein said mutations is/are at an amino acid positioncorresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169,S188, Q206, N212, N218, T254 or Q271 of SEQUENCE ID NO
 2. 55-60.(canceled)
 61. The enzyme of claim 44, wherein said one or moremutations correspond to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L,E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A or Q271E.
 62. Theenzyme of claim 44, comprising a mutation at both the positionscorresponding to N218 and M50.
 63. The enzyme of claim 44, comprisingmutations at the amino acid position corresponding to S3C and Q206C,wherein the cysteins at the positions corresponding to position 3 andposition 206 form a disulphur bridge.
 64. (canceled)
 65. The enzyme ofclaim 44, comprising at least six mutations, wherein said mutationscorrespond to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S,G169A, S188P, Q206C, N212G, N218S, T254A or Q271E.
 66. The enzyme ofclaim 65, wherein the enzyme comprises the mutations corresponding toQ2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P,Q206C, N212G, N218S, T254A or Q271E of SEQUENCE ID NO
 2. 67. The enzymeof claim 44, comprising one or more mutations at the amino acid positioncorresponding to N62, G100, S125, L126, G127, P129, N155, Y217, N218 orM222 of SEQUENCE ID NO
 2. 68. The enzyme of claim 67, comprising amutation at the position corresponding to M222 of SEQUENCE ID NO
 2. 69.The enzyme of claim 68, wherein said mutation at the positioncorresponding to M222 is M222G, M222P, M222N, M222E, M222Q or M222A. 70.The enzyme of claim 44, comprising a mutation at the amino acid positioncorresponding to Y217 of SEQUENCE ID NO
 2. 71. The enzyme of claim 70,wherein the mutation at the amino acid position corresponding to Y217 isY217L, Y217N, Y217E, Y217G, Y217F, Y217A, Y217S or Y217H.
 72. The enzymeof claim 71, wherein the mutation at amino acid position correspondingto Y217 is Y217F, Y217G or Y217H.
 73. The enzyme of claim 72, whereinthe enzyme comprises mutations at the amino acid positions correspondingto M222 and Y217, wherein the mutations are: M222P and Y217H; M222P andY217G; M222G and Y217F; or M222G and Y217G.
 74. The enzyme of claim 73,wherein said mutations are M222G and Y217F.
 75. The enzyme of claim 44,comprising at least one mutation selected from the group of mutations atan amino acid position corresponding to Y104, I107, L126, S101, G102,G127, G128, L135 or P168 of SEQUENCE ID NO
 2. 76. The enzyme of claim75, wherein said mutation is at an amino acid position corresponding toY104F, Y104S, I107V, I107A, L135N, L135S, L135D or L135A. 77-85.(canceled)